Tn-MUC4 binding polypeptides and uses thereof

ABSTRACT

The present inventors have demonstrated that circulating auto-antibodies to cancer antigens hold promise as specific and sensitive biomarkers for the early detection of cancer. The present invention thus relates to methods of detecting cancer in a sample, comprising utilising a glycopeptide bait derived from human mucins with different cancer-associated O-glycans. Detected antibodies were demonstrated as glycopeptide specific, and it could be discriminated between e.g. colorectal cancer patients and healthy individuals. The inventors have also developed monoclonal antibodies based on the identified glycopeptides epitope baits, and demonstrated differential expression of the relevant target antigens. The invention thus, in a lock and key-based manner includes both glycopeptides and antibody tools for early detection of cancer, as well as methods of using the same for in situ visualisation and treatment of specific cancer types.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to and is a divisional of U.S.patent application Ser. No. 13/505,827 filed on Jul. 16, 2012, now U.S.Pat. No. 9,588,121, issued Mar. 7, 2017, titled METHOD FOR EARLYDETECTION OF CANCER, which was a National Stage entry of InternationalPatent Application No. PCT/DK2010/050297 filed on Nov. 5, 2010, titledMETHOD FOR EARLY DETECTION OF CANCER, and which claims priority to U.S.Provisional Patent Application No. 61/259,023 filed on Nov. 6, 2009,titled METHOD FOR EARLY DETECTION OF CANCER, which are herebyincorporated by reference in their entirety.

All patent and non-patent references cited in the present applicationare hereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to methods for detecting and treatingcancer using multiple O-glycosylated mucin peptides, fragments andvariants, in addition to antibodies binding to said glycosylatedpeptides. In particular the invention relates to a method of detectingcancer, including but not limited to colorectal cancer, pancreaticcancer, breast cancer, oral cancer, gastric cancer, esophageal cancer,cholangiocarcinoma, ovarian cancer, lung cancer, renal cancer, prostatecancer, hepatocellular carcinoma, testis cancer, basal cell cancer,squamous cell cancer, malignant melanoma, bladder cancer, endometrialcancer and cervix cancer.

BACKGROUND OF INVENTION

Colorectal cancer develops in a multistep process that arises fromgenetic or epigenetic alterations (Vogelstein, Fearon et al. 1988). Mostcolorectal cancers can be treated by removal of early malignant lesions(Selby, Friedman et al. 1992; Etzioni, Urban et al. 2003), but despitethis colorectal cancer remains the second most common cause of cancerdeath in the United States (Jemal, Murray et al. 2005; Hospital 2006).Current screening techniques include fecal occult blood, double contrastbarium enema, sigmoid- and colonoscopy, as well as computed tomographiccolonography (Whitlock, Lin et al. 2008). Although these procedures areimportant in the early detection of colorectal cancer with significantimpact on mortality, they are complicated with low compliance and highcost (Ahlquist, Sargent et al. 2008; Whitlock, Lin et al. 2008). Withthe aim of facilitating surveillance and identification of high-riskpopulations, genomics and proteomics have elucidated many new potentialbiomarkers (Garcea, Sharma et al. 2003). Such efforts to develop asimple reliable non-invasive screening test for early detection ofcolorectal cancer had thus far been unsuccessful, mostly because theiruse in clinical practice has been hampered by lack of specificity andsensitivity (Kim, Yu et al. 2008). Thus, new biomarkers for the earlydetection of colorectal cancer are needed.

Circulating auto-antibodies serve as serological biomarkers with longcirculation time. They are the result of the inherent amplified functionof the immune system mirroring cancer specific structures not governedby self-tolerance (Anderson and LaBaer 2005). Several methodologies havebeen used to detect such auto-antibodies to cancer associated antigens(Stockert, Jager et al. 1998; Pereira-Faca, Kuick et al. 2007) (Mintz,Kim et al. 2003), (Chen, Scanlan et al. 1997; Stockert, Jager et al.1998; Jager, Stockert et al. 1999; Sugita, Wada et al. 2004), (Scanlan,Chen et al. 1998; Scian, Carchman et al. 2008) (Liu, Zhang et al. 2008).None of these technologies take post-translational modifications intoaccount.

WO 2008/040362 (Clausen et al) discloses a number of glycosylationpatterns of the MUC1 peptide VTSAPDTRPAPGSTAPPAHG but does not provideany hint as to the connection between the Core3 glycan(GlcNAcβ1-3GalNAc-α-Ser/Thr) in relation to detecting cancer.

U.S. Pat. No. 6,465,220 (Hassan et al) discloses a method ofglycosylating a MUC1 acceptor peptide, but does not provide anyinformation as how to use these glycopeptides for detecting colorectalcancer.

WO 1999/034824 (Karsten et al) discloses a tumour vaccine based on asynthetic MUC1 derived peptide PDTRPAP glycosylated on the Threonineresidue.

US 2003 0232399 (Robertson et al) discloses a method of detecting theimmune response of a mammal to circulating tumour marker proteins mainlyassociated with breast cancer, including MUC1, p53, c-erbB2, Ras, c-myc,BRCA1, BRCA2, PSA, APC and CA125. Robertson et al does not disclose thespecific glycopeptides of the present invention or specific antibodiesagainst these peptides.

US 2007 0240236 (Xia et al), also published as WO2008/147405, disclosesuse of an O-glycan composition (e.g., mucins) to prevent or treatinflammatory bowel diseases or gastrointestinal tumours. O-glycancompounds such as a mucin are proposed for the prevention and treatmentof a gastrointestinal cancer, such as colorectal cancer. However, Xia etal does not provide any solution to how to diagnose cancer at an earlystage by using the antibody-glycopeptide lock-and-key concept of thepresent invention.

WO 2006138275 (Wang et al) discloses compositions and methods fortreating, characterizing and diagnosing cancer, including CRC but do notmention the specific glycopeptides of the present invention.

Robbe-Masselot et al. (2009) J. Proteome Res. 8(2):702-11 discussesexpression of a core 3 disialyl-Le(x) hexasaccharide in human colorectalcancers as a potential marker of malignant transformation in colon. Thefindings of Robbe-Masselot et al. relates to MUC2. There is nodisclosure in this paper of the specific glycopeptides or antibodies ofthe present invention.

Thus the current state of the art does not provide a solution to theneed for new biomarkers for the early detection of cancer such ascolorectal cancer.

SUMMARY OF INVENTION

As a solution to the need for biomarkers for the early detection ofdisease such as, but not limited to cancer the present inventors havefound that specific changes in post-translational modifications, such asglycosylation pattern of the translated proteins are useful in detectingdisease. In relation to cancer, these changes also provide recognizablepatterns that, when suitably detected, can be used to discriminatebetween cancer and other diseases or disorders within the same tissue(s)and/or organ(s) that presents with the same or similar symptoms, forexample colorectal cancer and inflammatory bowel disease of thegastrointestinal tract.

By combining at least two glycosylated peptides, such as O-glycosylatedmucin it is possible at an early disease stage, to discriminate betweenautoantibodies resulting from cells suffering from different kinds ofdisorders and diseases. The method can also be reversed, by using anantibody to detect a glycosylated mucin peptide which may have been shedfrom a cell surface mucin as a consequence of disease in said cell. Inthis manner the antibody and the glycopeptide epitope may be consideredas plug and socket.

Thus in a first aspect, the present invention relates to a method fordetecting cancer, said method comprising

-   -   (i) contacting a sample from a sample host with        -   (a) at least two different mucin peptides,        -    wherein said at least two different mucin peptides            individually consists of an amino acid sequence comprising            at least 5 consecutive amino acid residues, and        -    wherein at least one of said at least 5 amino acid residues            are Serine or Threonine, and        -    wherein at least one of said Serine or Threonine residues            is O-glycosylated by a glycan independently selected from            the group consisting of:        -    Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr),        -    Tn (GalNAc-α-Ser/Thr),        -    STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr),        -    Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr))        -    Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and        -    ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr),        -    or,        -   (b) a monoclonal antibody, wherein said antibody is capable            of recognising at least one of the O-glycosylated mucin            peptides of (a),        -    and    -   (ii) removing unbound sample, and    -   (iii) qualitatively and/or quantitatively characterising the        bound material, wherein        -   (a) antibodies or antigen binding fragments of antibodies,            bound to said O-glycosylated peptides, or        -   (b) O-glycosylated peptide bound to said antibody    -   is indicative of cancer in the sample host.

The method is applicable both when using one or more peptides as well astwo, three, four or more different O-glycosylated peptides for detectingautoantibodies specific to different peptides, or an antibodycross-reacting in a known manner with known O-glycosylated mucinpeptides. The method has been demonstrated to be particularly useful indetecting colorectal cancer (CRC).

Thus, in another aspect, the present invention relates to a method fordetecting colorectal cancer, said method comprising

-   -   (i) contacting a sample from a sample host with        -   (a) one or more different mucin peptides,        -    wherein said one or more different mucin peptides            individually consists of an amino acid sequence comprising            at least 5 consecutive amino acid residues, and        -    wherein at least one of said at least 5 amino acid residues            are Serine or Threonine, and        -    wherein at least one of said Serine or Threonine residues            is O-glycosylated by a glycan independently selected from            the group consisting of:        -    Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr),        -    Tn (GalNAc-α-Ser/Thr),        -    STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr),        -    Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr))        -    Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and        -    ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr),        -    or,        -   (b) a monoclonal antibody, wherein said antibody is capable            of recognising at least one of the O-glycosylated mucin            peptides of (a),        -    and    -   (ii) removing unbound sample, and    -   (iii) qualitatively and/or quantitatively characterising the        bound material, wherein        -   (a) antibodies or antigen binding fragments of antibodies,            bound to said O-glycosylated peptides, or        -   (b) O-glycosylated peptide bound to said antibody    -   is indicative of colorectal cancer in the sample host.

In another aspect the invention relates to a method for detectingcancer, said method comprising

-   -   (i) contacting a sample from a sample host with        -   (a) one or more different mucin peptides,        -    wherein said one or more different mucin peptides            individually consists of an amino acid sequence comprising            at least 5 consecutive amino acid residues, and        -    wherein at least one of said at least 5 amino acid residues            are Serine or Threonine, and        -    wherein at least one of said Serine or Threonine residues            is O-glycosylated by a glycan independently selected from            the group consisting of:        -    Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr),        -    Tn (GalNAc-α-Ser/Thr),        -    STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr),        -    Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr))        -    Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and        -    ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr),        -    or,        -   (b) a monoclonal antibody, wherein said antibody is capable            of recognising at least one of the O-glycosylated mucin            peptides of (a),        -    and    -   (ii) removing unbound sample, and    -   (iii) qualitatively and/or quantitatively characterising the        bound material, wherein        -   (a) antibodies or antigen binding fragments of antibodies,            bound to said O-glycosylated peptides, or        -   (b) O-glycosylated peptide bound to said antibody    -   is indicative of cancer in the sample host.

While the method described herein above is particularly useful indetecting cancer, it may in principle be used to detect any disease witha similar disease pattern.

Thus in a further aspect, the present invention relates to a method fordetecting a disease in a sample host wherein said disease ischaracterised in that autoantibodies are produced by the individualsuffering from the disease, said method comprising

-   -   (i) contacting a sample from said sample host with        -   (a) at least two different mucin peptides,        -    wherein said at least two different mucin peptides            individually consists of an amino acid sequence comprising            at least 5 consecutive amino acid residues, and        -    wherein at least one of said at least 5 amino acid residues            are Serine or Threonine, and        -    wherein at least one of said Serine or Threonine residues            is O-glycosylated by a glycan independently selected from            the group consisting of:        -    Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr),        -    Tn (GalNAc-α-Ser/Thr),        -    STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr),        -    Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr))        -    Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and        -    ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr),        -    or,        -   (b) a monoclonal antibody, wherein said antibody is capable            of recognising at least one of the O-glycosylated mucin            peptides of        -    (a),        -    and    -   (ii) removing unbound sample, and    -   (iii) qualitatively and/or quantitatively characterising the        bound material, wherein        -   (a) antibodies or antigen binding fragments of antibodies,            bound to said O-glycosylated peptides, or        -   (b) O-glycosylated peptide bound to said antibody    -   is indicative of disease in the sample host.

An object of the present invention is to use the general method definedabove for detecting specific disorders characterised in thatautoantibodies are produced by the individual suffering from saiddisease. One such disease is cancer. An individual afflicted with cancersignificantly benefits from an early stage diagnosis. Accordingly, inone aspect the present invention relates to a method for detectingcancer, said method comprising

(i) contacting a sample with one or more O-glycosylated mucin peptides,wherein said peptide is selected from the group consisting of:

PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID N07), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24),PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25)andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), or a fragment of said peptides, or variants of saidpeptides in which variants any amino acid has been changed to adifferent amino acid, provided that no more than 5 of the amino acidresidues in the sequence are so changed, and wherein the asterisk (*)indicates a potential O-glycosylation site, wherein the optional glycanis independently selected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr) and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

In a further aspect the present invention concerns a method fordetecting cancer, said method comprising

(i) contacting a sample with one or more mucin peptides, wherein saidpeptide is selected from the group consisting of:

a) MUC4 Tn selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

b) a MUC4 non glycosylated mucin peptide selected from the groupconsisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ IDNO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a Tnglycosylated mucin peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

c) a Tn glycosylated MUC4 peptide selected from the group consisting ofPMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

d) an all-Tn MUC4 peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*TTS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylationsite, wherein the glycan is Tn (GalNAc-α-Ser/Thr), or

e) a recombinant MUC4 Tn having the sequencePMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

f) a MUC1Tn/STn/Core3 glycosylated or MUC4 Tn glycosylated mucin peptideselected from the group consisting ofVT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG,PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),

andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30),

wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), or

g) a MUC1 STn and a MUC4 selected from the group consisting ofVT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), andwherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr),

and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

The peptides of the invention may be produced either by digesting fulllength or truncated mucin polypeptides or by automated peptide synthesisand optionally glycosylated as described herein below. These methods forpeptide synthesis and glycosylation are known to those skilled in theart.

In principle the present invention is applicable to any glycosylatedpeptide epitope and a corresponding antibody, acting in a lock andkey-manner, for the detection of various disorders and diseases. Thepresent inventors have found that the efficiency of detection isparticularly good when two or more different glycosylated peptides areused for the detection of disease. Thus, in one aspect the inventionrelates to a method for detecting a disease in an individual whereinsaid disease is characterised in that autoantibodies are produced by theindividual suffering from the disease, said method comprising

(i) contacting a sample with at least two glycosylated peptides, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides are indicative ofdisease or disorder in the sample host.

While the present invention is applicable to any optionally glycosylatedpeptide and a corresponding antibody, for the detection of variousdisorders and diseases, the present inventors have also identified anumber of specific mucin based peptides that are useful in detectingcolorectal cancer.

Accordingly, in a further aspect, the present invention relates to amethod for detecting colorectal cancer in a host organism, said methodcomprising

(i) contacting a sample from said host organism with one or moreO-glycosylated mucin peptides, wherein said peptide(s) is/are selectedfrom the group consisting of: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24),PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25)andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), or a fragment of said peptides, or variants of saidpeptides in which variants any amino acid has been changed to adifferent amino acid, provided that no more than 5 of the amino acidresidues in the sequence are so changed, and wherein the asterisk (*)indicates a potential O-glycosylation site, wherein the optional glycanis independently selected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

As demonstrated in table II of the present application, and as discussedherein above, it may be useful to utilise one or more, such as twodifferent O-glycosylated peptides for detecting colorectal cancer, andfor distinguishing between colorectal cancer and inflammatory boweldisease.

Thus, in one aspect the present invention relates to a method fordetecting colorectal cancer in a sample host, said method comprising

(i) contacting a sample from said sample host with one or more mucinpeptides, wherein said peptide is selected from the group consisting of:

a) MUC4 Tn selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

b) a MUC4 non glycosylated mucin peptide selected from the groupconsisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ IDNO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a MUC4 Tnglycosylated mucin peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

c) a Tn glycosylated MUC4 peptide selected from the group consisting ofPMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

d) an all-Tn MUC4 peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylationsite, wherein the glycan is Tn (GalNAc-α-Ser/Thr), or

e) a recombinant MUC4 Tn having the sequencePMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

f) a MUC1Tn/STn/Core3 glycosylated or MUC4 glycosylated mucin peptideselected from the group consisting ofVT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG,PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),

andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30),

wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), or

g) a MUC1 STn and a MUC4 selected from the group consisting ofVT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), andwherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr),

and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

In principle, any suitable O-glycosylated peptide can be used fordetecting autoantibodies binding to this glycosylated peptide epitope.It is preferred that the peptide epitope comprises at least 5 amino acidresidues.

Thus, in one aspect the invention relates to a method for detectingcolorectal cancer in a host organism, said method comprising

(i) contacting a sample from said host organism with at least twodifferent O-glycosylated peptides, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to the peptides of step (i) areindicative of disease or disorder in the host organism.

In one aspect the present invention thus relates to a method fordetecting colorectal cancer in a host organism, said method comprising

(i) contacting a sample from said host organism with one or moreO-glycosylated peptides, wherein said peptide comprises at least 5consecutive amino acid residues of a mucin selected from the groupconsisting of MUC4 (SEQ ID NO: 1), MUC1 (SEQ ID NO: 2), MUC1 VariantCT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 VariantY, MUC1 Variant ZD, MUC2 (SEQ ID NO: 26), MUC3A, MUC3B, MUC4, MUC5AC(SEQ ID NO: 27), MUC5B, MUC6 (SEQ ID NO: 28), and MUC7 (SEQ ID NO: 29),MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17,MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variantthereof, wherein said variant is at least 70% identical to said at least5 consecutive amino acid residues of said mucin, or a naturallyoccurring fragment or variant of said mucin, wherein said variant is atleast 70% identical to said at least 5 consecutive amino acid residuesof said mucin, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

The peptide can be any peptide sharing a significant sequence identitywith the peptides specified herein above, or a peptide sharing secondaryor tertiary structure with the present peptides. Thus, in a furtheraspect the present invention relates to a method for detectingcolorectal cancer in a host organism, said method comprising

(i) contacting a sample from said host organism with one or moreO-glycosylated mucin peptides, wherein said peptide comprises, or saidpeptides comprise, at least 5 consecutive amino acid residues of amucin, or a fragment or variant thereof, wherein said variant is atleast 70% identical, such as at least 75% identical to, e.g. at least80% identical to, such as at least 85% identical to, e.g. at least 90%identical to, such as at least 95% identical to, e.g. at least 98%identical to, such as at least 99% identical to said at least 5consecutive amino acid residues of said mucin, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe host organism.

The MUC1 peptides of the invention have been found to be useful indetecting disease, and in particular colorectal cancer. Thus in oneaspect the invention relates to a method for detecting colorectalcancer, said method comprising

(i) contacting a sample with one or more O-glycosylated MUC1 peptidesand optionally at least one second mucin peptide wherein theO-glycosylated MUC1 peptides are selected from the group consisting ofVTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) and wherein said at least onesecond mucin peptide optionally is/are selected from the groupconsisting of: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is independentlyselected from the group consisting of Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr) and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

As discussed above, the method requires both parts of the lock-and-keymechanism to be present. The method thus also relates to use of theantibodies of the invention to detect glycopeptides epitopes binding tosaid antibodies.

Thus in another aspect, the present invention relates to a method fordetecting cancer, said method comprising

(i) contacting a sample with an antibody capable of recognising one ormore O-glycosylated mucin peptides, wherein said peptide comprises atleast 5 consecutive amino acid residues of a mucin, or a fragment orvariant thereof, wherein said variant is at least 70% identical, such asat least 75% identical to, e.g. at least 80% identical to, such as atleast 85% identical to, e.g. at least 90% identical to, such as at least95% identical to, e.g. at least 98% identical to, such as at least 99%identical to said at least 5 consecutive amino acid residues of saidmucin, and

(ii) removing unbound sample and/or unbound antibody and/or unboundantibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

As with the peptides defined herein above, also the antibodies of thepresent invention may be used for detecting a disease associated withshedding of antibody target epitopes such as peptides. Therefore, in oneaspect, the present invention also relates to a method for detecting agastrointestinal disease in a host organism wherein said disease ischaracterised in that O-glycosylated mucin peptides are shed from thediseased host and secreted in the gastrointestinal tract of the sampleorganism suffering from the disease, said method comprising

(i) contacting a sample from said host organism with one or moreantibodies capable of recognising said O-glycosylated mucin peptides,and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to the antibodies of step (i) areindicative of disease or disorder in the host organism.

In another aspect the invention relates to a method for detecting adisease such as a gastrointestinal disease, in a host organism whereinsaid disease is characterised in that O-glycosylated mucin peptides areshed from the diseased cells of the host into an extracellular volume,such as secreted into, the lumen of the bladder, milk ducts of thebreast, lumen of the uterus, the vagina, into pancreatic fluid, intoascites fluid, onto bronchiolar surface of the lung, ductal surfaces ofthe prostate, lumen of the seminiferous tubules, the oesophagus or thegastrointestinal tract of the sample organism suffering from thedisease, said method comprising

(i) contacting a sample from said host organism with one or moreantibodies capable of recognising said O-glycosylated mucin peptides,and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to the antibodies of step (i) areindicative of disease or disorder in the host organism.

The present inventors have found glycopeptide epitopes associated withcolorectal cancer and antibodies useful in detecting said glycopeptides.Therefore, in a further aspect, the present invention relates to amethod for detecting colorectal cancer, said method comprising

(i) contacting a sample with at least one antibody, wherein saidantibody is capable of recognising a glycosylated mucin peptide selectedfrom the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3),PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO:11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ IDNO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21),PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ IDNO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), andPTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25) or a fragment of saidpeptides, or variants of said peptides in which variants any amino acidhas been changed to a different amino acid, provided that no more than 5of the amino acid residues in the sequence are so changed, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

The present inventors have raised several antibodies specific fordifferent glycosylated parts of the mucin proteins. Accordingly, in oneaspect the present invention relates to a method for detecting cancer,said method comprising

(i) contacting a sample with at least two different antibodies, whereinsaid antibodies are capable of recognising two different glycosylatedmucin peptides, wherein said peptide comprises at least 5 consecutiveamino acid residues of a mucin selected from the group consisting ofMUC4 (SEQ ID NO:1), MUC1 (SEQ ID NO: 2), MUC2 (SEQ ID NO: 26), MUC5AC(SEQ ID NO: 27), MUC6 (SEQ ID NO: 28), and MUC7 (SEQ ID NO: 29) or anaturally occurring fragment or variant of said mucin, wherein saidvariant is at least 70% identical to said at least 5 consecutive aminoacid residues of said mucin,

and

(ii) removing unbound sample and/or unbound antibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In another aspect, the present invention relates to a method fordetecting colorectal cancer, said method comprising (i) contacting asample with a polyclonal antibody serum wherein said polyclonal antibodyis capable of recognising at least two different O-glycosylated mucinpeptides, wherein said peptides comprises at least 5 consecutive aminoacid residues selected from the group consisting of MUC1 Variant CT58,MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y,MUC1 Variant ZD,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7,MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17,MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variantthereof, wherein said variant is at least 70% identical to said at least5 consecutive amino acid residues of said mucin, and (ii) removingunbound sample and/or unbound antibody), and (iii) qualitatively and/orquantitatively characterise the bound material, wherein peptides boundto said antibodies indicate cancer in the sample host.

The antibodies of the present invention have been raised againstdifferent parts of glycosylated mucin polypeptides, Thus in one aspect,the present invention relates to a monoclonal antibody or an antigenbinding fragment of said antibody, wherein said antibody or the antigenbinding fragment of said antibody is capable of specifically recognisinga mucin glycopeptide as defined herein above.

In one aspect the invention relates to a method for detecting cancer,said method comprising (i) contacting a sample with at least one firstand at least one second antibody, wherein said first antibody is capableof recognising a first mucin peptide selected from the group consistingof the O-glycosylated MUC1 peptides VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn), and wherein said secondantibody is capable of recognising another mucin peptide selected fromthe group consisting of:

PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is independentlyselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In one aspect the invention relates to a method for detecting cancer,said method comprising (i) contacting a sample with at least oneantibody, wherein said antibody is capable of recognising a glycosylatedmucin peptide, wherein said peptide comprises at least 5 consecutiveamino acid residues of a mucin selected from the group consisting ofMUC4 (SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), or a fragment or variantthereof, wherein said variant is at least 70% identical to said at least5 consecutive amino acid residues of said mucin selected from the groupconsisting of MUC4 (SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), and

(ii) removing unbound sample and/or unbound antibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In one aspect the present invention relates to a method for detectingcolorectal cancer, said method comprising

(i) contacting a sample with at least one antibody, wherein saidantibody is capable of recognising a glycosylated mucin peptide selectedfrom the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3),PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO:11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ IDNO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21),PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ IDNO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), andPTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25) or a fragment of saidpeptides, or variants of said peptides in which variants any amino acidhas been changed to a different amino acid, provided that no more than 5of the amino acid residues in the sequence are so changed, and

(ii) removing unbound sample and/or unbound antibody

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In one aspect the present invention relates to a monoclonal antibody oran antigen binding fragment of said antibody, wherein said antibody orthe antigen binding fragment of said antibody is capable of specificallyrecognising a mucin glycopeptide as defined herein above. Therecognition may be tested in a binding assay such as a Biacore assay (GEHealthcare, General Electric Company). These methods are well known tothose skilled in the art.

The method used by the present inventors for raising antibodies is alsopart of the invention. Thus, in one aspect, the present inventionrelates to a method for producing the antibody defined herein above,said method comprising the steps of:

-   -   i) providing a host organism,    -   ii) immunizing the host organism with an O-glycosylated mucin as        defined herein above, and    -   iii) obtaining said antibody.

The present invention is applicable to a subject undergoing therapy,such as surgery. In surgery the glycopeptide epitopes identified by thepresent inventors can be targeted with antibodies of the inventionwherein the antibodies are conjugated to a visualization label, such asa fluorescent label. A variety of visualisation labels may be conjugatedto the antibodies by methods well known to those skilled in the art, forexample labels that are suitable for visualisation by X-rayfluoroscopes, Computer Assisted X-ray Tomography (CAT), PositronEmission Tomography (PET) and Nuclear Magnetic Resonance Imaging devices(NMRI). The epitopes can thus be visualised prior to or during surgerywith the effect of more efficiently removing e.g. cancer tumours.Accordingly, in one aspect, the present invention relates to a methodfor detecting a cancer tumour in a patient undergoing therapy and/orexamination, said method comprising the steps of:

(i) administering to an area of the patient, the antibody as definedherein above, or an antigen binding fragment of said antibody optionallylabelled with a visualisation probe (i.e. a visualisation probeconjugated to the antibody of the invention) so that theantibody-visualisation probe complex acts as an imaging agent

(ii) removing unbound antibodies, and

(iii) detecting antibodies bound to glycopeptide epitopes as definedherein above, wherein labelling indicates presence of a tumour.

The glycopeptides of the invention have been used to construct a devicefor detecting disease, in particular cancer and especially colorectalcancer. Thus, in one aspect, the present invention relates to a devicecomprising a plurality of glycosylated peptides attached to a surface,wherein at least a part of the peptides are selected from the peptidesdefined herein above.

As discussed above, the glycopeptides and the antibodies of theinvention act in a lock-and-key manner. Thus, in one aspect, the presentinvention relates to a device comprising a plurality of antibodiescovalently attached to a surface, wherein at least a part of theantibodies are selected from the antibodies as defined herein, i.e.including but not limited to the antibodies selected from the groupconsisting of MAb 5C10, MAb 3C9, MAb 4D9, MAb 6C11 and MAb 6E3.

In one aspect the invention relates to a device comprising at least twodifferent O-glycosylated peptides conjugated to a surface, wherein theat least two different peptides are selected from the peptides definedherein above.

In an analogous aspect, the invention relates to a device comprising aplurality of different antibodies conjugated to a surface, wherein oneor more of the antibodies is/are selected from the antibodies definedherein.

In one aspect the device is used in a method of identifyingglycopeptides such as glycopeptides associated with disease, said methodcomprising contacting the device defined herein above with a sample froma host organism. In a further aspect, when a plurality of antibodieshave been conjugated to the surface of the device in place ofglycopeptide constructs, said device can be used in a method to identifythe corresponding O-glycosylated peptides associated with disease, saidmethod comprising contacting said device with a sample from a hostorganism.

In another aspect, the device is used in a method of identifyingglycopeptides associated with disease, said method comprising contactingsaid device with a sample from a host organism. When glycopeptides havebeen conjugated to the device, said device can be used in a method ofidentifying antibodies associated with disease, said method comprisingcontacting said device with a sample from a host organism.

In one aspect, the present invention relates to a mucin peptide asdefined herein above for use as a medicament, in particular for use in amethod of treatment of cancer. Said mucin peptide used in said method oftreatment of cancer can e.g. include immunisation of an individual byadministering to said individual the glycosylated mucin peptide. Themedical use also applies to the other part of the lock-and-keyinvention. Thus, in this aspect the present invention relates to amethod of immunising an individual, by administering to said individualan antibody of the invention.

The antibodies of the present invention can be used for detectingO-glycosylated peptides associated with disease. The O-glycosylatedpeptides in turn may be used to detect the autoantibodies alsoassociated with disease. Thus in one aspect the present invention thusrelates to a method of detecting an O-glycosylated peptide or theantibody as defined herein above, said method comprising conjugating animaging agent to said peptide or antibody and detecting said imagingagent with any technique suitable thereto.

The peptides of the invention can be used individually or conjugated toa suitable object which may simplify the detection process. In oneaspect the invention thus relates to a device comprising a plurality ofglycosylated peptides conjugated to a surface, wherein at least a partof the peptides are selected from the peptides defined herein above. Inanother aspect the invention relates to a device comprising a pluralityof antibodies conjugated to a surface, wherein at least a part of theantibodies are selected from the antibodies defined herein above.

The antibodies and the corresponding peptide epitopes of the presentinvention are not limited to methods for detecting disease, but can alsobe used in methods for treating disease. In one aspect the inventionrelates to a mucin peptide as defined herein above for use as amedicament. In one aspect the invention also relates to a mucin peptideas defined in herein for use in a method of treatment of cancer. In oneaspect the invention relates to use of one or more antibodies as definedherein above for the manufacture of a medicament for the treatment ofcancer. For example the antibodies of the invention can be used aspassive immunization agents, singly or in combination to target cells ofparticular cancers. The antibodies can optionally be conjugated with oneor more toxins that, alone or in combination, can kill cells targeted bythe antibodies. Passive immunotherapy where antibodies binds to tumourcell surfaces and thereby facilitates tumour-killing by effector cellsare also part of the invention. In one embodiment the antibodies of theinvention are conjugated with a radioactive molecule capable of killinga tumour cell after binding of the antibody:radioactive molecule complexto the tumour cell surface.

In one aspect, the present invention relates to a method for identifyinga disease (e.g. cancer such as colorectal cancer) associated withshedding of O-glycosylated peptides or peptide fragments, said methodcomprising the steps of:

-   -   (i) selecting potential target polypeptides containing potential        O-glycosylation sites, and    -   (ii) producing recombinant fragments covering specific areas of        interest from each potential target, and/or    -   (iii) producing synthetic peptides covering specific areas of        interest from each potential target, and    -   (iv) in vitro glycosylate the fragments of (ii) and/or (iii)        using recombinant glycosyltransferases    -   (v) purifying the fragments of (iv), and    -   (vi) characterizing the purified products of (v), and    -   (vii) printing of non-glycosylated and glycosylated targets,    -   (viii) screening the printed targets of (vii) with sera from a        potentially diseased sample host and    -   (ix) screening the printed targets of (vii) with sera from a        healthy sample host as control,    -   wherein the presence of auto-antibodies bound to the printed        targets of (viii) indicates disease in the potentially diseased        sample host.

While the present invention mainly is directed to a method of detectingcancer, it can also be applied for monitoring the efficacy or effect ofradiotherapy and/or chemotherapy. Thus in one aspect the presentinvention relates to method for monitoring the efficacy of achemotherapy and/or radiotherapy treatment, said method comprising thesteps of

-   -   (i) contacting a sample from a sample host with at least two        different mucin peptides, wherein said at least two different        mucin peptides individually consists of an amino acid sequence        comprising at least 5 consecutive amino acid residues, and        wherein at least one of said at least 5 amino acid residues are        Serine or Threonine, and    -    wherein at least one of said Serine or Threonine residues is        O-glycosylated by a glycan independently selected from the group        consisting of:    -    Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr),    -    Tn (GalNAc-α-Ser/Thr),    -    STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr),    -    Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr))    -    Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and    -    ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and    -   (ii) removing unbound sample, and    -   (iii) qualitatively and/or quantitatively characterising the        bound material, wherein antibodies or antigen binding fragments        of antibodies, bound to said O-glycosylated peptides, is        indicative of efficient/successful chemotherapy and/or        radiotherapy treatment of the sample host.

DESCRIPTION OF THE DRAWINGS

FIG. 1A to 1E-3: The O-glycopeptide microarray platform. FIG. 1A:Biosynthetic pathways of the initial steps in the most common mucin-typeO-glycans. Each pathway can be further elongated and modified (notshown). Candidate glycosyltransferases responsible for each biosyntheticstep are indicated. FIG. 1B: Graphical overview of a number of studiedmucins explored in the present work. Demonstrated are signal sequences(SP), tandem repeat domains (TR), and transmembrane domains (TM). MUC1and MUC4 are membrane associated mucins, while MUC2, MUC5AC, MUC6, andMUC7 are secreted mucins. FIG. 1C: Position of the glycopeptides on thearray. Peptides were printed in three different concentration and fourreplicates. FIG. 1D-1 to 1-D3: Fluorescent image of glycopeptide arraywith bound monoclonal antibodies specific for the peptide tandem repeatof MUC1 (5E10) (FIG. 1D-1) and carbohydrate structures Tn (GalNAcα)(FIG. 1D-2) and STn (NeuAcα6GalNAcα) (FIG. 1D-3). Monoclonal antibodieswere detected with Cy3-conjugated anti-mouse IgG overlaid onto theprinted glycopeptide array, washed, and scanned for fluorescence.Quantification of antibody recognition profiles is shown for eachfluorescent image with the use of the mean value of the 4 replicates isshown in FIG. 1E-1 to 1E-1E3, respectively.

FIG. 2A to 2F: IgG auto-antibodies to MUC1 glycopeptides Quantificationof the antibody responses for glycopeptide targets in each individualshown in a 3-dimensional bar graph for healthy individuals (n=50) (FIG.2A), inflammatory bowel disease patients (UC or CD in active orremission phase; n=39) (FIG. 2B), and colorectal cancer patients(Pre-treatment (n=58), Stage I-IV, (I n=23, II n=5, III n=23, IV n=7))(FIG. 2C). Columns indicate the IgG reactivity in serum from one patient(y-axis) expressed as relative fluorescence units (z-axis) for eachlisted compound x-axis). MUC1 glycopeptide targets were predominantlyrecognised by colorectal cancer patients as demonstrated in DOT-PLOTdiagram illustrating the level of IgG autoantibody reactivity of eachindividual towards glycoforms of MUC1 (DOT-PLOT only depict results fromselected boxed target glyco-peptides from 3-dimensional bar graph). Dotsin DOT-PLOT represent the relative fluorescent value for individualcontrols and patients. Healthy individuals demonstrated none or very lowreactivity against Tn- and STn-MUC1 glycoforms (FIG. 2D). A significantnumber of patients with Inflammatory Bowel Disease (IBD) hadauto-antibodies against Core-3-MUC1 compared with healthy individuals(FIG. 2E). In contrast only few IBD patients had auto-antibodies againstTn- and STn-MUC1. In colorectal cancer IgG auto-antibodies were detectedagainst Tn-, STn, and Core-3-MUC1 in a large proportion of the patients(FIG. 2F), with 15STn-MUC1 being the best marker to distinguish thecancer group from the healthy group. For Tn- and STn MUC1 similarreactivity was seen between recombinant (rMUC1) and synthetic peptides(MUC1) covering the MUC1 tandem repeat. Statistical details are shown inFIG. 3.

FIG. 3A-1 to 3G-2: Sialyl-Tn-MUC1 auto-antibodies predict the presenceof colorectal cancer. FIG. 3A-1 to 3A-2: Focused DOT-PLOT diagrampresenting auto-antibody reactivity against Core3-MUC1 (FIG. 3A-1) andSTn-MUC1 (FIG. 3A-2). Each dot represents the relative fluorescent unitfor each individual in the three groups of patients examined (i) Healthyindividuals (blood donors), (ii) Colorectal cancer patients(Pre-treatment, Stage I-IV), (iii) Inflammatory bowel disease patients.56.7% of patients with established colorectal cancer have significantlyincreased level of auto-antibodies to STn-MUC1 (above 3 standarddeviation over the mean value of the healthy control group. FIG. 3B-1 to3B-2: ROC curve for serum IgG antibodies to Core3-MUC1 (FIG. 3B-1) andSTn-MUC1 (FIG. 3B-2). Area under the curve is 0.7944 and 0.8774respectively. The value of specificity is plotted as 1-specificity onthe x-axis. FIG. 3C-1 to 3C-2: DOT-PLOT diagram of auto-antibodyreactivity in colorectal cancer patients without (stage I-III) or withmetastasis (stage IV). Patients with metastasis have lower activity ofauto-antibodies against Core3-MUC1 (FIG. 3C-1) and STn-MUC1 (FIG. 3C-2).See FIG. 8 for further characterization of the sub-groups. FIG. 3D-1 to3D-2 Analysis of O-glycopeptide specificity of detected auto-antibodies.FIG. 3D-1 to 3G-2: Fluorescent images of the array showing thereactivity pattern of the same cancer patient serum, afterpre-incubation with 40 μg/mL of unglycosylated MUC1 peptide (FIG. 3D-1to 3D-2), 15Core-3-MUC1 (FIG. 3E-1 to 3E-2), 15STn-MUC1 (FIG. 3F-1 to3F-2), or Core-3-MUCSAC (FIG. 3G-1 to 3G-2) as control. FIGS. 3D-2,3E-2, 3F-2, and 3G-2: Quantification of the STn-MUC1 and Core-3-MUC1signal depicted in FIGS. 3D-1, 3E-1, 3F-1, and 3G-1, respectively. Thecancer specific serum reactivity to STn-MUC1 and Core-3-MUC1 wasinhibited by 40 ug/mL of the respective MUC1 glycopeptides STn-MUC1 andCore-3-MUC1. No cross-reaction was observed.

FIG. 4A to 4C-3: Auto-antibodies of the IgA subclass targeting MUC4preferentially identifies colorectal cancer patients. FIG. 4A: Graphicaloverview of MUC4 and localization of recombinant MUC4 construct andshort peptides covering the recombinant fragment and tandem repeatdomain. FIG. 4B-1 to 4B-4: 3-dimensional bar graph illustrating IgAauto-antibodies against MUC4 non glycosylated and glycosylated peptidesin healthy individuals (FIG. 4B-1), inflammatory bowel disease patients(FIG. 4B-2), colorectal cancer patients (FIG. 4B-3), and a mixedpopulation of breast, ovarian, and prostate cancer patients (FIG. 4B-4).FIG. 4C-1 to 4C-3: 3-dimensional bar graph Y-axis represents the numberof standard deviations (SD) above the mean of the values obtained withsera from healthy individuals in colorectal cancer patients (FIG. 4C-1),inflammatory bowel disease patients (FIG. 4C-2), and a mixed populationof breast, ovarian, and prostate cancer patients (FIG. 4C-3). The Y-axiscut-off level is 5 SD over the mean value of the controls.

FIG. 5A to 5F-2: Monoclonal antibodies to Core3-MUC1 and Tn-MUC4. FIG.5A: MAb to Core3-MUC1 (5C10) characterized by ELISA. Antigen doubledilution is presented on the x-axis. FIG. 5B-1: Microarraycharacterization of mAb to Core3-MUC1 (5C10) on the mucin array. FIG.5B-2: 2-Dimensional bar graph illustrating the relative fluorescentquantification of the array result. Specific reactivity was seen toCore3-MUC1 with Core3 structure in the PDTR sequence of MUC1 tandemrepeat. FIG. 5C: Monoclonal antibody to Tn-MUC4 (6E3) characterized byELISA with different recombinant mucin glycoproteins. FIG. 5D-1 to 5D-2:Mucin micro-array characterisation of mAb 6E3. Strongest activity wasseen to MUC4Tn. Less reactivity was seen toward MUC1Tn and MUC5ACTn.FIG. 5E: Polyclonal response from a mouse immunized with Tn-MUC4TR5analyzed by ELISA. Dilution of serum from eyebleed is presented on theX-axis. Glyco-peptide specific reactivity to Tn-MUC4TR5 was seen with noreaction to hapten Tn (on MUC2) or unglycosylated MUC4 peptide. FIG.5F-1 to 5F-2: Serum from mouse immunized with Tn-MUC4TR5 on a focusedMUC4 microarray. Reactivity was only seen to Tn-MUC4TR5 with noreactivity to non-glycosylated or other MUC4 Tn-glycopeptides.

FIG. 6A to 6B: FITC staining of tissue from colon. Sections of healthycolon, colon affected with inflammatory bowel disease, and colorectalcancer were stained with a panel of monoclonal antibodies for MUC1 (mAbHMHG2), Tn/STn-MUC1 (mAb 5E5) and Core3-MUC1 (mAb 5C10) (FIG. 6A), andMUC4, Tn-MUC4 (6E3 and 4D9) and Tn-MUC4-TR5 (FIG. 6B). Increasedstaining for Core3-MUC1 is seen with 5C10 in colorectal cancer and IBDcompared with healthy colon. Albeit MUC4 is present in small quantitiesin healthy colon, an increase in staining intensity and surfaceexpression is seen with a commercial MUC4 mAb, and the two Tn-MUC4antibodies (6E3 and 4D9) in colorectal cancer.

FIG. 7A to 7B-3: Production of cancer-associated glycopeptides andglycoproteins. FIG. 7A: Workflow from DNA construct to glycoprotein.Various construct designs were tested for optimal expression efficacy.Expression cultures were NiNTA and HPLC purified, analysed by CoomassiePAGE analysis and MALDI-TOF mass spectrometry analysis (FIG. 7B-1 to7B-3). Mucin constructs were expressed in pET28 with a N-terminal6xHIS-T7 tag. Purified recombinant mucin fragments were in vitroglycosylated using purified human recombinant GalNAc-transferases. MALDIdata for both naked purified recombinant protein and glycosylatedrecombinant protein (rMUC4+T2 and rMUC7+T2) is presented. The Tnglycoforms were further elongated to make cancer-associated glycoforms(Core-3, T and STn). Recombinant fragment of MUC2 (rMUC4) and MUC7(rMUC7) are shown as examples. Similar results were obtained with theremaining mucin fragments.

FIG. 8A to 8T: Quantification of IgG auto-antibodies against recombinantprotein MUC2 (FIG. 8A-8D), MUC4 (FIG. 8E-8H), MUCSAC (FIG. 81-8L), MUC6(FIG. 8M-8P) and MUC7 (FIG. 8Q-8T). Differential auto-antibody responsesto recombinant protein MUC2, MUC4, MUC5AC, MUC6 and MUC7 betweencolorectal cancer patients compared with healthy controls were notidentified.

FIG. 9A to 9B: DOT-PLOT diagram presenting auto-antibodies to Core3-MUC1(FIG. 9A) and STn-MUC1 (FIG. 9B) in the colorectal cancer patients. Thepatients are subdivided in accordance to the location and to the stageof the tumour.

FIG. 10A-1 to 10E-2: Purification and characterization ofauto-antibodies from cancer serum FIG. 10A-1 to 10A-2: IgGauto-antibodies from a colorectal cancer patient evaluated on the mucinarray. Auto-antibodies are seen to STn-MUC1, Core3-MUC1, and otherrecombinant mucins mainly with the Core-3 glycans. FIG. 10B-1 to 10B-2:Reactivity of IgG auto-antibodies after pre-absorption of Core-3-MUC1auto-antibodies by incubation of the serum with recombinant Core3-MUC1coupled to dynabeads via Histidine tag. The Core3-MUC1 signal is reducedover 90%, while STn-MUC1 and the other mucins carrying the Core3-glycansare unaffected. FIG. 10C-1 to 10C-2: Core-3-MUC1 specific IgGauto-antibodies eluted from the Core3-MUC1-dynabeads incubated withserum. Specific reactivity to Core3-MUC1 was recovered. FIG. 10D-1 to10D-2: IgM antibodies in serum from the same cancer patient. Reactivitywas seen towards different proteins especially carrying Core-3. FIG.10E-1 to 10E-2: IgM auto-antibodies eluted from Core3-MUC1-dynabeadsafter incubation with the same cancer serum. Eluted IgM auto-antibodiesreacted with all carriers of Core-3.

FIG. 11A-1 to 11B-3: Characterization of Mab 5C10 to Core-3-MUC1

FIG. 11A-1 to 11A-3 MAb 5C10 characterized by MUC1 microarray. Specificreactivity was seen to Core3-MUC1 with Core3 structure in the PDTRsequence of MUC1 tandem repeat. FIG. 11B-1 to 11B-3: MAb 5C10characterized on the MUC1 microarray after elongation of Core-3-MUC1with galactose (Galb3GlcNAcb3GalNAcα) by the glycosyl-transferasesβ3Gal-T5. The reactivity to Core3-MUC1 where completely diminished afterelongation.

FIG. 12A to 12D: IgA autoantibodies to MUC1 Glycopeptides.

DOT-PLOT diagram presenting IgA auto-antibody reactivity against MUC1(FIG. 12A), Tn-MUC1 (FIG. 12B), STn-MUC1 (FIG. 12C), and Core-3-MUC1(FIG. 12D) in healthy individuals (blood donors), Colorectal cancerpatients, Inflammatory bowel disease patients.

FIG. 13A to 13C: Specificity of IgA autoantibodies to Tn-MUC4 Selectiveinhibition of autoantibodies to recombinant Tn-MUC4. Bar diagram withquantifications of the Tn-MUC1, Tn-MUC2, Tn-MUC4, Tn-MUC5AC, Tn-MUC6,and 7 reactivity inhibited with 40 μg/mL of unglycosylated MUC4 (FIG.13A), to-MUC4 (FIG. 13B) or Tn-MUC2 (FIG. 13C). RV: relative value.

FIG. 14: Antibody-Dependent Cellular Cytotoxicity (ADCC) MCF7 The bargraph presenting the antibody-dependent cellular cytotoxocity of PBMC(Peripheral blood mononuclear cells) with 6E3, a negativ controlantibody, and PBMC alone. Results from effector cell/tumor ratio of 25:1and 50:1 are shown. On the y-axis is the percentage of the specificrelease of 51Cr. The error bars indicates standard deviation.

FIG. 15A to 15R: DOT-PLOT diagram presenting autoantibody reactivityagainst p53 derived peptides p53-4 (FIG. 15A), p53-5 (FIG. 15B), p53-9(FIG. 15C), p53-10 (FIG. 15D), p53-14 (FIG. 15E), p53-25 (FIG. 15F),p53-26 (FIG. 15G), p53-27 (FIG. 15H), p53-34 (FIG. 15L), p53-39 (FIG.15J), p53-41 (FIG. 15K), p53-42 (FIG. 15L), p53-44 (FIG. 15M), 53-45(FIG. 15N), p53-43 (FIG. 15O), p53-58 (FIG. 15P), p53-59 (FIG. 15Q),p53-78 (FIG. 15R). Each dot represents one individual in the groupsblood donors (n=29), 53 healthy controls (n=53), colorectal cancerpatients (n=58), breast cancer patients (n=26), ovarian cancer patients(n=12), and benign diseases (n=20, mixture of patients with benignbreast tumors, benign ovarian tumors, and prostate hyperplasia). Therelative fluorescence units (RFU) are presented on the y-axis.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Disorder: The term ‘disorder’ used herein refers to a disease or medicalproblem, and is an abnormal condition of an organism that impairs bodilyfunctions, associated with specific symptoms and signs. It may be causedby external factors, such as invading organisms, or it may be caused byinternal dysfunctions, such as impaired catecholamine production ortransport. In particular, a disorder as used herein is cancer.

Glycan: The term glycan as used herein refers to a polysaccharide oroligosaccharide. Glycan may also be used to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid, or aproteoglycan. Glycans usually consist solely of O-glycosidic linkages ofmonosaccharides. For example, cellulose is a glycan (or morespecifically a glucan) composed of beta-1,4-linked D-glucose, and chitinis a glycan composed of beta-1,4-linked N-acetyl-D-glucosamine. Glycanscan be homo or heteropolymers of monosaccharide residues, and can belinear or branched.

Glycans of the invention are listed below. N-Acetylgalactosamine(GalNAc), is an amino sugar derivative of galactose(2-Acetamido-2-deoxy-D-galactose) with the molecular formula C₈H₁₅NO₆.N-Acetylglucosamine (GlcNAc), is an amino sugar derivative of glucose(2-Acetamido-2-deoxy-D-glucose) with the molecular formula C₈H₁₅NO₆.

Galactosamine is a hexosamine derived from galactose with the molecularformula C₆H₁₃NO₅.

Sialic acid is a generic term for N-acetylneuraminic acid (Neu5Ac orNANA). The amino group can be varied with either an acetyl or glycolylgroup but other modifications have been described. The hydroxylsubstituents may vary considerably: acetyl, lactyl, methyl, sulfate, andphosphate groups have been found.

STn or sialyl-Tn antigen: (Neu5Acα2-6GalNAc-Ser/Thr)

Tn or Tn antigen: (GalNAc-Ser/Thr)

Core-2: Galβ3(GlcNAcβ6)GalNAc-Ser/Thr)

Core-3: GlcNAcβ3GalNAc-Ser/Thr)

Core-4: GlcNAcβ3(GlcNAcβ6)GalNAc-Ser/Thr)

ST or sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr)

Pharmaceutical composition: or drug, medicament or agent refers to anychemical or biological material, compound, or composition capable ofinducing a desired therapeutic effect when properly administered to apatient. Some drugs are sold in an inactive form that is converted invivo into a metabolite with pharmaceutical activity. For purposes of thepresent invention, the terms “pharmaceutical composition” and“medicament” encompass both the inactive drug and the active metabolite.

Polypeptide: The term “polypeptide” as used herein refers to a moleculecomprising at least two amino acids. The amino acids may be natural orsynthetic. “Oligopeptides” are defined herein as being polypeptides oflength not more than 100 amino acids. The term “polypeptide” is alsointended to include proteins, i.e. functional biomolecules comprising atleast one polypeptide; when comprising at least two polypeptides, thesemay form complexes, be covalently linked or may be non-covalentlylinked. The polypeptides in a protein can be glycosylated and/orlipidated and/or comprise prosthetic groups.

Protein: The term ‘protein’ used herein refers to an organic compound,also known as a polypeptide, which is a peptide having at least, andpreferably more than two amino acids. The generic term amino acidcomprises both natural and non-natural amino acids any of which may bein the ‘D’ or ‘L’ isomeric form.

Methods for Detecting Disease

The present inventors have developed a chemo-enzymatic approach toproduce libraries of disease-associated O-glycopeptides such ascancer-associated O-glycopeptides printed on a microarray platformallowing high through-put detection of auto-antibodies to theO-glycopeptidome. Based on this the inventors have created an expandedmucin glycopeptide array to identify disease-associated glycopeptidetargets. The inventors have for example created an expanded mucinglycopeptide array useful in identifying cancer associated glycopeptidetargets being specific for detection of colon cancer specific autologousantibodies.

While the invention is useful in the detection of autoantibodies andcorresponding glycopeptide epitopes recognised by said autoantibodies,the invention is also applicable to any glycopeptide associated withdisease or disorder. The data provides clear support for the utility ofthis approach and provides guidance for the person skilled in the art tofurther improve specificity and sensitivity of the targets provided. Asa solution to the need for biomarkers for the early detection of diseasesuch as, but not limited to cancer the present inventors have found thatspecific changes in post-translational modifications, such asglycosylation pattern of the translated proteins are useful in detectingdisease. In relation to cancer, these changes also provide recognizablepatterns that may be used to discriminate between cancer such ascolorectal cancer and inflammatory disease of the gastrointestinaltract.

In principle the present invention is applicable to any glycosylatedpeptide epitope and a corresponding antibody, acting in a lock andkey-manner, for the detection of various disorders and diseases. Thepresent inventors have found that the efficiency of detection isparticularly good when two or more different glycosylated peptides areused for the detection of disease.

In one aspect the present invention concerns a method for detecting adisease in a host organism wherein said disease is characterised in thatautoantibodies are produced by the individual suffering from thedisease, said method comprising

(i) contacting a sample from said host organism with at least twodifferent O-glycosylated peptides, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to the peptides of step (i) areindicative of disease or disorder in the host organism.

In one embodiment, the at least two different O-glycosylated peptidesare mucin peptides.

In one embodiment of the above method, the disease is cancer.

In a further embodiment the cancer is selected from the group consistingof colorectal cancer, breast cancer, oral cancer, gastric cancer,esophageal cancer, pancreatic cancer, cholangiocarcinoma, ovariancancer, lung cancer, renal cancer, prostate cancer, hepatocellularcarcinoma, testis cancer, basal cell cancer, squamous cell cancer,malignant melanoma, bladder cancer, endometrial cancer and cervixcancer.

The glycopeptide targets in MUC1(Tn-MUC1, STn-MUC1, Core-3-MUC1) foundby the inventors, are recognised by auto-antibodies in colorectal cancerpatients. In addition the inventors have found epitopes in Tn-MUC4(GalNAc-al MUC4), which allowed the inventors to distinguish betweencolorectal cancer patients and healthy individuals and patients withinflammatory bowel disease, respectively.

Thus in one embodiment the cancer detected or treated by the presentinvention is colorectal cancer.

The novel concept for the simple and non-invasive identification ofpatients with e.g. colorectal cancer is a significant advantage overcurrent screening techniques by its use of immunological amplifiedsignals present at early stages of the disease. The strategy can beperformed alone or in combination with current screening efforts such acolonoscopy. Conventional endoscopic screening is limited due to lack ofmolecular specificity and because only anatomical changes are revealedthrough a macroscopic view of the surface mucosa. Therefore, flat ordepressed neoplasms are difficult to detect using endoscopic methods,and especially patients with chronic inflammatory bowel disease are atincreased risk of developing malignancy due to undetected dysplasticlesions. There is thus a need for improved methods for detecting earlychanges in high-risk individuals. New approaches includes the use ofnovel imaging techniques as well as combining endoscopic efforts withfluorescent imaging with either non-specific or specific dyes (Hsiung,Hardy et al. 2008). Another perspective is to combine these approacheswith serum markers, which could serve as indicators for the presence ofdisease and hence warrant further examination by existing techniques. Inthis way serum marker strategies could be complementary to anatomicaldata provided by imaging techniques.

In summary, the inventors have provided enabling support for a novelO-glycopeptide array for the sensitive detection of disease-associatedauto-antibodies, such as cancer associated auto-antibodies. The datafurthermore provides clear support for the utility of this approach andprovides guidance for the person skilled in the art to further improvespecificity and sensitivity of the targets provided. The invention thusprovides a method for screening patients routinely for possible presenceof a disease, imaging agents for in situ disclosure and definition ofthe volume of diseased tissue, and highly specific therapeutic agents totreat the disease.

In relation to colorectal cancer, the inventors have found that patientsafflicted with colorectal cancer selectively generate antibodiesrecognizing Tn-MUC1, STn-MUC1 and Tn-MUC4, while auto-antibodies againstCore-3-MUC1 were generated in both colorectal cancer and inflammatorybowel patients. Combining the cancer associated glycoforms of MUC1 andMUC4 as target antigens resulted in detection of 82% of the cancerpatients with a specificity of 95% (See table II).

The inventors have found that the predominant epitope identified byauto-antibodies against STn and Core-3-MUC1 is located in the -GSTAP-motif of the MUC1 tandem repeat sequence carrying two glycans, while afew patients have additional immunoreactivity with glyco-peptides withone glycan in either T in -VTS- or T in -PDTR (See FIG. 2 and Table II).

The rather large difference in the auto-antibody levels between patientsis noteworthy and indicates important biological variations inauto-antibody production and responses in cancer patients. First,interpersonal variations in the amount of expressed antigen(glycan-MUC1) could vary in colorectal cancer. However, using Tn/STn-MUC1 specific antibodies the inventors found that 92% (23/25) of theexamined patients express Tn/STn-MUC1. In contrast only 50% of thepatients have STn-MUC1 antibodies. Additional explanations thereforeexist for the variations in auto-antibody levels. Among these, thesubject's ability to recognise and present STn- and Core3-MUC1 could beimportant. In order to test if patients with high levels ofauto-antibodies corresponded to patients in which peripheral T cellresponse could be identified, the inventors isolated T-cells from 15selected colon cancer patients. Furthermore, variance in local stromalfactors such as the secretion of TGF beta among other factors are knownto down-regulate the immune-response to cancer targets causingimmunological escape (Tinder, Subramani et al. 2008). This is a possibleexplanation to the lack of selected immunological reaction such asanti-STn-MUC1 in some individuals. Finally, many of these patients couldbe immuno-compromised due to the progression of their disease. In thisrespect the inventors detected a deterioration of both the Core-3-MUC1and STn-MUC1 response in later stage cancers with liver metastasis (FIG3C). This could reflect the immuno-compromised state of the patients orchanges in expression of the antigen. Alternatively, the decrease inserum antibodies in late stage cancers is due to chelation ofcirculating antibodies by large tumour and metastasis mass along withthe possibility of immune-complex formation, which would render theantibodies undetectable. In summary, the results show that the method ofthe present invention is useful in detecting cancer at an early stage.

The immunogenic nature of MUC1 provides an explanation for MUC1glycopeptide responses in cancers other than gastrointestinal cancers,such as breast, ovarian, and prostate cancer. In these patients,however, only few have circulating MUC1 auto-antibodies.

In one embodiment the cancer detected or treated by the presentinvention is colorectal cancer.

In one embodiment the at least two different O-glycosylated peptides ofthe method of the present invention as defined herein above comprises atleast 5 consecutive amino acid residues of a mucin selected from thegroup consisting of MUC1 Variant CT58, MUC1 Variant CT80, MUC1 VariantSEC, MUC1 Variant X, MUC1 Variant Y, MUC1 Variant ZD,MUC2, MUC3A, MUC3B,MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13,MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or afragment or variant thereof, wherein said variant is at least 70%identical to said at least 5 consecutive amino acid residues of saidmucin. In one embodiment the O-glycosylated peptide of the method of thepresent invention is not a MUC2 peptide.

In one embodiment the at least 5 consecutive amino acid residues of amucin is/are selected from the group consisting of MUC1 Variant CT58,MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y,MUC1 Variant ZD,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7,MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17,MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variantthereof, wherein said variant is at least 70% identical to said at least5 consecutive amino acid residues of said mucin are selected from thegroup consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3),PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO:11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ IDNO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ IDNO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23),VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), andPTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25), or a fragment of saidpeptides, or variants of said peptides in which variants any amino acidhas been changed to a different amino acid, provided that no more than 5of the amino acid residues in the sequence are so changed.

In one embodiment of the method of the present invention as definedherein, the at least two O-glycosylated peptides are a first and asecond O-glycosylated mucin peptide wherein the first O-glycosylatedmucin peptide is selected from the group consisting ofVTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn),

and the second O-glycosylated mucin peptide is selected from the groupconsisting of: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is independentlyselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In principle the peptides of the invention may be glycosylated by anyphysiological O-glycan at a potential O-glycosylation site. Thus, in oneembodiment the optional glycan is Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr).In another embodiment the optional glycan is Tn (GalNAc-α-Ser/Thr). Inyet another embodiment the optional glycan is STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr). In yet another embodiment the optionalglycan is Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)). In anotherembodiment the optional glycan is a Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr). In yet another embodiment theoptional glycan is ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In one embodiment of the present invention the at least two differentO-glycosylated peptides are at least three different O-glycosylatedpeptides selected from the group consisting of:VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24),PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25),PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is independentlyselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr)

In one embodiment of the present invention the at least two differentO-glycosylated peptides are at least four different O-glycosylatedpeptides selected from the group consisting of:VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24),PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25),PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is independentlyselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)), Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In one embodiment of the present invention the at least two differentO-glycosylated peptides are five or more different O-glycosylatedpeptides selected from the group consisting of:VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24),PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25),PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is independentlyselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)), Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr)

In one embodiment the five or more different O-glycosylated peptides aresix or more O-glycosylated peptides, such as seven or moreO-glycosylated peptides, for example eight or more O-glycosylatedpeptides, such as nine or more O-glycosylated peptides, for example tenor more O-glycosylated peptides, such as eleven or more O-glycosylatedpeptides, for example twelve or more O-glycosylated peptides, such as 13or more O-glycosylated peptides.

An object of the present invention is to use the general method definedabove for detecting specific disorders characterised in thatautoantibodies are produced by the individual suffering from saiddisease. One such disease is cancer. An individual afflicted with cancersignificantly benefits from an early stage diagnosis.

Accordingly, in one aspect the present invention relates to a method fordetecting cancer, said method comprising

(i) contacting a sample with one or more O-glycosylated mucin peptides,wherein said peptide is selected from the group consisting of:PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24),PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25)andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), or a fragment of said peptides, or variants of saidpeptides in which variants any amino acid has been changed to adifferent amino acid, provided that no more than 5 of the amino acidresidues in the sequence are so changed, and wherein the asterisk (*)indicates a potential O-glycosylation site, wherein the optional glycanis independently selected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)), Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr) and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

The glycosylated peptides of the present invention preferably comprisesat least 5 amino residues. In one aspect the present invention relatesto a method for detecting cancer, said method comprising

(i) contacting a sample with one or more O-glycosylated mucin peptides,wherein said peptide comprises at least 5, such as at least 8, e.g. atleast 10, such as at least 15 consecutive amino acid residues of a mucinselected from the group consisting of MUC4 (SEQ ID NO: 1) and/or MUC1(SEQ ID NO: 2), or a fragment or variant thereof, wherein said variantis at least 70%, for example at least 75%, such as at least 80%, forexample at least 85%, such as at least 90%, for example at least 95%,such as at least 98%, for example at least 99% identical to said atleast 5 consecutive amino acid residues of said mucin selected from thegroup consisting of MUC4 (SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

The peptides of the invention may be produced either by digesting fulllength or truncated mucin polypeptides or by automated peptidesynthesis. The peptides are then optionally glycosylated. These methodsare known to those skilled in the art.

In one aspect the present invention relates to a method for detectingcancer, said method comprising

(i) contacting a sample with one or more mucin peptides, wherein saidpeptide is selected from the group consisting of:

a) MUC4 Tn selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

b) a MUC4 non glycosylated mucin peptide selected from the groupconsisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ IDNO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a Tnglycosylated mucin peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

c) a Tn glycosylated MUC4 peptide selected from the group consisting ofPMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

d) an all-Tn MUC4 peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylationsite, wherein the glycan is Tn (GalNAc-α-Ser/Thr), or

e) a recombinant MUC4 Tn having the sequencePMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

f) a MUC1Tn/STn/Core3 glycosylated or MUC4 glycosylated mucin peptideselected from the group consisting ofVT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG,PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),

andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30),

wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), or

g) a MUC1 STn and a MUC4 selected from the group consisting ofVT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), andwherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr),

and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

In one embodiment the cancer detected by the method of the presentinvention is selected from the group consisting of colorectal cancer,breast cancer, oral cancer, gastric cancer, esophageal cancer,pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer,renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer,basal cell cancer, squamous cell cancer, malignant melanoma, bladdercancer, endometrial cancer and cervix cancer.

In one embodiment the detected cancer is colorectal cancer.

In one aspect the present invention relates to a method for detectingcolorectal cancer in a host organism, said method comprising

(i) contacting a sample from said host organism with one or moreO-glycosylated mucin peptides, wherein said peptide is selected from thegroup consisting of: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24),PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25)andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), or a fragment of said peptides, or variants of saidpeptides in which variants any amino acid has been changed to adifferent amino acid, provided that no more than 5 of the amino acidresidues in the sequence are so changed, and wherein the asterisk (*)indicates a potential O-glycosylation site, wherein the optional glycanis independently selected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

As demonstrated in table II of the present application, and as discussedherein above, it may be useful to utilise one or more, such as twodifferent O-glycosylated peptides for detecting colorectal cancer, andfor distinguishing between colorectal cancer and inflammatory boweldisease.

Thus, in one aspect the present invention relates to a method fordetecting colorectal cancer in a sample host, said method comprising

(i) contacting a sample from said sample host with one or more mucinpeptides, wherein said peptide is selected from the group consisting of:

a) MUC4 Tn selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

b) a MUC4 non glycosylated mucin peptide selected from the groupconsisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ IDNO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a Tnglycosylated mucin peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

c) a Tn glycosylated MUC4 peptide selected from the group consisting ofPMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

d) an all-Tn MUC4 peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*TTS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylationsite, wherein the glycan is Tn (GalNAc-α-Ser/Thr), or

e) a recombinant MUC4 Tn having the sequencePMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

f) a MUC1Tn/STn/Core3 glycosylated or MUC4 glycosylated mucin peptideselected from the group consisting ofVT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG,PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),

andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30),

wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), or

g) a MUC1 STn and a MUC4 selected from the group consisting ofVT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), andwherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr),

and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

In a further aspect the invention relates to a method for detectingcolorectal cancer in a host organism, said method comprising

(i) contacting a sample from said host organism with at least twodifferent O-glycosylated peptides, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to the peptides of step (i) areindicative of disease or disorder in the host organism.

In principle, any suitable O-glycosylated peptide can be used fordetecting auto-antibodies binding to this glycosylated peptide epitope.It is preferred that the peptide epitope comprises at least 5 amino acidresidues.

In one aspect the present invention thus relates to a method fordetecting colorectal cancer in a host organism, said method comprising

(i) contacting a sample from said host organism with one or moreO-glycosylated peptides, wherein said peptide comprises at least 5consecutive amino acid residues of a mucin selected from the groupconsisting of MUC4 (SEQ ID NO: 1), MUC1 (SEQ ID NO: 2), MUC1 VariantCT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 VariantY, MUC1 Variant ZD, MUC2 (SEQ ID NO: 26), MUC3A, MUC3B, MUC4, MUC5AC(SEQ ID NO: 27), MUC5B, MUC6 (SEQ ID NO: 28), and MUC7 (SEQ ID NO: 29),MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17,MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variantthereof, wherein said variant is at least 70% identical to said at least5 consecutive amino acid residues of said mucin, or a naturallyoccurring fragment or variant of said mucin, wherein said variant is atleast 70% identical to said at least 5 consecutive amino acid residuesof said mucin, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

In one embodiment said O-glycosylated mucin peptide is selected from thegroup consisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ IDNO: 22) and PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23).

In another embodiment said O-glycosylated mucin peptide is a MUC4selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is selected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In another embodiment said O-glycosylated mucin peptide is a MUC4 Tnselected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr).

In another embodiment said O-glycosylated mucin peptide is a MUC4 nonglycosylated or Tn glycosylated mucin selected from the group consistingof PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ IDNO: 22) and PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theglycan is Tn (GalNAc-α-Ser/Thr).

In another embodiment said O-glycosylated mucin peptide is a MUC4fragment Tn selected from the group consisting ofPMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16), GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is a Tn(GalNAc-α-Ser/Thr).

In another embodiment said O-glycosylated mucin peptide is an all-TnMUC4 peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr).

In another embodiment the at least two different O-glycosylated mucinpeptides are a first O-glycosylated mucin peptide selected from thegroup consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3),PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO:11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ IDNO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21),PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ IDNO: 23) and a second O-glycosylated mucin peptide selected from thegroup consisting of VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) respectively, wherein said firstO-glycosylated mucin peptide is glycosylated on one or more Thr or Serresidues and wherein said glycosylation is

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

In another embodiment, wherein the one or more O-glycosylated mucinpeptides and/or the two or more O-glycosylated mucin peptides as definedanywhere in the present application, are a first O-glycosylated mucinpeptide and a second O-glycosylated mucin peptide wherein said firstO-glycosylated mucin peptide is a MUC1 O-glycosylated mucin peptide andwherein said second O-glycosylated mucin peptide is a MUC4O-glycosylated mucin peptide.

In another embodiment said MUC1 O-glycosylated mucin peptide is aMUC1STn and/or MUC1Tn and/or MUC1Core3 O-glycosylated mucin peptideselected from the group consisting ofVT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG, and whereinsaid MUC4 O-glycosylated mucin peptide is selected from the groupconsisting of PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQID NO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the glycan is independently and optionallyselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In one aspect the present invention relates to a method for detectingcolorectal cancer, said method comprising:

(i) contacting a sample with one or more O-glycosylated MUC1 peptidesand optionally at least one second mucin peptide wherein theO-glycosylated MUC1 peptides are selected from the group consisting ofVTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) and wherein said at least onesecond mucin peptide optionally is/are selected from:

PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ ID NO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 30), and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

The peptide can be any peptide sharing a significant sequence identitywith the peptides specified herein above, or a peptide sharing secondaryor tertiary structure with the present peptides. Thus, in a furtheraspect the present invention relates to a method for detectingcolorectal cancer in a host organism, said method comprising

(i) contacting a sample from said host organism with one or moreO-glycosylated mucin peptides, wherein said peptide comprises, or saidpeptides comprise, at least 5 consecutive amino acid residues of amucin, or a fragment or variant thereof, wherein said variant is atleast 70% identical, such as at least 75% identical to, e.g. at least80% identical to, such as at least 85% identical to, e.g. at least 90%identical to, such as at least 95% identical to, e.g. at least 98%identical to, such as at least 99% identical to said at least 5consecutive amino acid residues of said mucin, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe host organism.

All mucin peptides defined herein above may occur as tandem repeats.Thus in one embodiment, the glycopeptide of the present invention is atandem repeat peptide. In one embodiment the number of repeats is two.In another embodiment the number of repeats is three. In yet anotherembodiment the number of repeats is four. In yet another embodiment thenumber of repeats is five or more, such as six, for example seven, suchas eight, for example nine, such as ten, for example eleven, such as 12,for example 13, such as 14, for example 15, such as 16, for example 17,such as 18, for example 19, such as 20, for example 21, such as 22, forexample 23, such as 24, for example 25, such as 26, for example 27, suchas 28, for example 29, such as 30, for example 31, such as 32, forexample 33, such as 34, for example 35, such as 36, for example 37, suchas 38, for example 39, such as 40, for example 41, such as 42, forexample 43, such as 44, for example 45, such as 46, for example 47, suchas 48, for example 49, such as 50 or more.

The term variant as used herein should be understood as functionalequivalent and can be used interchangeably. In one preferred embodimentof the invention there is also provided variants of the mucinglycopeptides and variants or fragments thereof. When beingpolypeptides, variants are determined on the basis of their degree ofidentity or their homology with a predetermined amino acid sequence,said predetermined amino acid sequence being one of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO: 29 and SEQ ID NO: 30, or, when the variant is afragment, a fragment of any of the aforementioned amino acid sequences,respectively.

Accordingly, variants preferably have at least 75% sequence identity,for example at least 80% sequence identity, such as at least 85%sequence identity, for example at least 90% sequence identity, such asat least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with thepredetermined sequence.

Sequence identity is determined in one embodiment by utilising fragmentsof the glycosylated peptides comprising at least 5 contiguous aminoacids and having an amino acid sequence which is at least 80%, such as85%, for example 90%, such as 95%, for example 99% identical to theamino acid sequence of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO: 29 or SEQ ID NO: 30, respectively, wherein the percent identity isdetermined with the algorithm GAP, BESTFIT, or FASTA in the WisconsinGenetics Software Package Release 7.0, using default gap weights.

Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2: 482, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.(U.S.A.) 85: 2444, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by inspection, and the best alignment (i.e., resulting in thehighest percentage of homology over the comparison window) generated bythe various methods is selected.

The term “sequence identity” means that two peptide or polypeptidesequences are identical (i.e., on an amino acid to amino acid basis)over the window of comparison. The term “percentage of sequenceidentity” is calculated by comparing two optimally aligned sequencesover the window of comparison, determining the number of positions atwhich the identical amino acid (e.g., A, G, T, S etc.) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The terms “substantialidentity” as used herein denotes a characteristic of a peptide orpolypeptide sequence, wherein the peptide or polypeptide sequencecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a predeterminedsequence over a comparison window of at least 20 amino acid positions,wherein the percentage of sequence identity is calculated by comparingthe predetermined sequence to the peptide or polypeptide sequence whichmay include deletions or additions which total 20 percent or less of thepredetermined sequence over the window of comparison. The predeterminedsequence may be a subset of a larger sequence, for example, as a segmentof the full-length mucin polypeptide sequences illustrated herein.

Furthermore, a degree of identity of amino acid sequences is a functionof the number of identical amino acids at positions shared by the aminoacid sequences. A degree of homology or similarity of amino acidsequences is a function of the number of amino acids, i.e. structurallyrelated, at positions shared by the amino acid sequences.

An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity, with one of thepeptide or polypeptide sequences of the present invention. The term“substantial identity” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 80 percent sequence identity, preferably atleast 90 percent sequence identity, more preferably at least 95 percentsequence identity or more (e.g., 99 percent sequence identity).Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine, a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

Additionally, variants are also determined based on a predeterminednumber of conservative amino acid substitutions as defined herein below.Conservative amino acid substitution as used herein relates to thesubstitution of one amino acid (within a predetermined group of aminoacids) for another amino acid (within the same group), wherein the aminoacids exhibit similar or substantially similar characteristics.

Within the meaning of the term “conservative amino acid substitution” asapplied herein, one amino acid may be substituted for another within thegroups of amino acids indicated herein below:

-   i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His,    Asn, Gln, Ser, Thr, Tyr, and Cys,)-   ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu,    Ile, Phe, Trp, Pro, and Met)-   iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu,    Ile)-   iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)-   v) Amino acids having aromatic side chains (Phe, Tyr, Trp)-   vi) Amino acids having acidic side chains (Asp, Glu)-   vii) Amino acids having basic side chains (Lys, Arg, His)-   viii) Amino acids having amide side chains (Asn, Gln)-   ix) Amino acids having hydroxy side chains (Ser, Thr)-   x) Amino acids having sulphur-containing side chains (Cys, Met),-   xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,    Thr)-   xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and-   xiii) Hydrophobic amino acids (Leu, Ile, Val)

Accordingly, a variant or a fragment thereof according to the inventionmay comprise, within the same variant of the sequence or fragmentsthereof, or among different variants of the sequence or fragmentsthereof, at least one substitution, such as a plurality of substitutionsintroduced independently of one another.

It is clear from the above outline that the same variant or fragmentthereof may comprise more than one conservative amino acid substitutionfrom more than one group of conservative amino acids as defined hereinabove.

The addition or deletion of at least one amino acid may be an additionor deletion of from preferably 2 to 250 amino acids, such as from 10 to20 amino acids, for example from 20 to 30 amino acids, such as from 40to 50 amino acids. However, additions or deletions of more than 50 aminoacids, such as additions from 50 to 100 amino acids, addition of 100 to150 amino acids, addition of 150-250 amino acids, are also comprisedwithin the present invention. The deletion and/or the additionmay—independently of one another—be a deletion and/or an addition withina sequence and/or at the end of a sequence.

The polypeptide fragments according to the present invention, includingany functional equivalents thereof, may in one embodiment comprise lessthan 250 amino acid residues, such as less than 240 amino acid residues,for example less than 225 amino acid residues, such as less than 200amino acid residues, for example less than 180 amino acid residues, suchas less than 160 amino acid residues, for example less than 150 aminoacid residues, such as less than 140 amino acid residues, for exampleless than 130 amino acid residues, such as less than 120 amino acidresidues, for example less than 110 amino acid residues, such as lessthan 100 amino acid residues, for example less than 90 amino acidresidues, such as less than 85 amino acid residues, for example lessthan 80 amino acid residues, such as less than 75 amino acid residues,for example less than 70 amino acid residues, such as less than 65 aminoacid residues, for example less than 60 amino acid residues, such asless than 55 amino acid residues, for example less than 50 amino acidresidues, such as less than 45 amino acid residues, for example lessthan 40 amino acid residues, such as less than 35 amino acid residues,for example less than 30 amino acid residues, such as less than 25 aminoacid residues, for example less than 20 amino acid residues, such asless than 15 amino acid residues, for example less than 12 amino acidresidues, such as less than 10 amino acid residues, for example lessthan 8 amino acid residues, such as less than 7 amino acid residues, forexample less than 6 amino acid residues, such as less than 5 amino acidresidues, for example less than 4 amino acid residues, such as less than3 amino acid residues.

“Functional equivalency” as used in the present invention is accordingto one preferred embodiment established by means of reference to thecorresponding functionality of a predetermined fragment of the sequence.

Functional equivalents or variants of a glycosylated mucin peptide willbe understood to exhibit amino acid sequences gradually differing fromthe preferred predetermined sequence, as the number and scope ofinsertions, deletions and substitutions including conservativesubstitutions increases. This difference is measured as a reduction inhomology between the preferred predetermined sequence and the fragmentor functional equivalent.

All fragments or functional equivalents of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO: 29 and SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68 and SEQ ID NO:69 are included within the scope of this invention,regardless of the degree of homology that they show to the respective,predetermined peptide sequences disclosed herein. The reason for this isthat some regions of the peptide are most likely readily mutatable, orcapable of being completely deleted, without any significant effect onthe binding activity of the resulting fragment.

The homology between amino acid sequences may be calculated using wellknown scoring matrices such as any one of BLOSUM 30, BLOSUM 40, BLOSUM45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70,BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.

Fragments sharing homology with fragments of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO: 29 and SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68 and SEQ ID NO:69, respectively, are to be considered as fallingwithin the scope of the present invention when they are preferably atleast about 90 percent homologous, for example at least 92 percenthomologous, such as at least 94 percent homologous, for example at least95 percent homologous, such as at least 96 percent homologous, forexample at least 97 percent homologous, such as at least 98 percenthomologous, for example at least 99 percent homologous with saidpredetermined fragment sequences, respectively. According to oneembodiment of the invention the homology percentages refer to identitypercentages.

Additional factors that may be taken into consideration when determiningfunctional equivalence according to the meaning used herein are i) theability of antisera to detect a peptide fragment according to thepresent invention, or ii) the ability of the functionally equivalentpeptide fragment to compete with the corresponding peptide in an assay,such as an inhibition assay. One method of determining a sequence ofimmunogenically active amino acids within a known amino acid sequencehas been described by Geysen in U.S. Pat. No. 5,595,915 and isincorporated herein by reference.

A further suitably adaptable method for determining structure andfunction relationships of peptide fragments is described by U.S. Pat.No. 6,013,478, which is herein incorporated by reference. Also, methodsof assaying the binding of an amino acid sequence to a receptor moietyare known to the skilled artisan.

In addition to conservative substitutions introduced into any positionof a preferred predetermined sequence, or a fragment thereof, it mayalso be desirable to introduce non-conservative substitutions in any oneor more positions of the glycopeptides of the invention.

A non-conservative substitution leading to the formation of afunctionally equivalent peptide fragment would for example i) differsubstantially in polarity, for example a residue with a non-polar sidechain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met) substituted for aresidue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, orGln or a charged amino acid such as Asp, Glu, Arg, or Lys, orsubstituting a charged or a polar residue for a non-polar one; and/orii) differ substantially in its effect on polypeptide backboneorientation such as substitution of or for Pro or Gly by anotherresidue; and/or iii) differ substantially in electric charge, forexample substitution of a negatively charged residue such as Glu or Aspfor a positively charged residue such as Lys, His or Arg (and viceversa); and/or iv) differ substantially in steric bulk, for examplesubstitution of a bulky residue such as His, Trp, Phe or Tyr for onehaving a minor side chain, e.g. Ala, Gly or Ser (and vice versa).

Variants obtained by substitution of amino acids may in one preferredembodiment be made based upon the hydrophobicity and hydrophilicityvalues and the relative similarity of the amino acid side-chainsubstituents, including charge, size, and the like. Exemplary amino acidsubstitutions which take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

In addition to the variants described herein, sterically similarvariants may be formulated to mimic the key portions of the variantstructure and that such compounds may also be used in the same manner asthe variants of the invention. This may be achieved by techniques ofmodelling and chemical designing known to those of skill in the art. Itwill be understood that all such sterically similar constructs fallwithin the scope of the present invention.

In a further embodiment the present invention relates to functionalvariants comprising substituted amino acids having hydrophilic values orhydropathic indices that are within +/−4.9, for example within +/−4.7,such as within +/−4.5, for example within +/−4.3, such as within+/−1-4.1, for example within +/−3.9, such as within +/−3.7, for examplewithin +/−3.5, such as within +/−3.3, for example within +/−3.1, such aswithin +/−2.9, for example within +/−2.7, such as within +/−2.5, forexample within +/−2.3, such as within +/−2.1, for example within +/−2.0,such as within +/−1.8, for example within +/−1.6, such as within +/−1.5,for example within +/−1.4, such as within +/−1.3 for example within+/−1.2, such as within +/−1.1, for example within +/−1.0, such as within+/−0.9, for example within +/−0.8, such as within +/−0.7, for examplewithin +/−0.6, such as within +/−0.5, for example within +/−0.4, such aswithin +/−0.3, for example within +/−0.25, such as within +/−0.2 of thevalue of the amino acid it has substituted.

The importance of the hydrophilic and hydropathic amino acid indices inconferring interactive biologic function on a protein is well understoodin the art (Kyte & Doolittle, 1982 and Hopp, U.S. Pat. No. 4,554,101,each incorporated herein by reference).

The amino acid hydropathic index values as used herein are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5) (Kyte & Doolittle, 1982).

The amino acid hydrophilicity values are: arginine (+3.0); lysine(+3.0); aspartate (+3.0.+−0.1); glutamate (+3.0.+−0.1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4) (U.S. Pat. No.4,554,101).

In addition to the peptidyl compounds described herein, stericallysimilar compounds may be formulated to mimic the key portions of thepeptide structure and that such compounds may also be used in the samemanner as the peptides of the invention. This may be achieved bytechniques of modelling and chemical designing known to those of skillin the art. For example, esterification and other alkylations may beemployed to modify the amino terminus of, e.g., a di-arginine peptidebackbone, to mimic a tetra peptide structure. It will be understood thatall such sterically similar constructs fall within the scope of thepresent invention.

Peptides with N-terminal alkylations and C-terminal esterifications arealso encompassed within the present invention. Functional equivalentsalso comprise glycosylated and covalent or aggregative conjugates formedwith the same or other peptide fragments, including dimers or unrelatedchemical moieties. Such functional equivalents are prepared by linkageof functionalities to groups which are found in fragment including atany one or both of the N- and C-termini, by means known in the art.

Functional equivalents may thus comprise fragments conjugated toaliphatic or acyl esters or amides of the carboxyl terminus, alkylaminesor residues containing carboxyl side chains, e.g., conjugates toalkylamines at aspartic acid residues; O-acyl derivatives of hydroxylgroup-containing residues and N-acyl derivatives of the amino terminalamino acid or amino-group containing residues, e.g. conjugates withfMet-Leu-Phe or immunogenic proteins. Derivatives of the acyl groups areselected from the group of alkyl-moieties (including C3 to C10 normalalkyl), thereby forming alkanoyl species, and carbocyclic orheterocyclic compounds, thereby forming aroyl species. The reactivegroups preferably are difunctional compounds known per se for use incross-linking proteins to insoluble matrices through reactive sidegroups.

Covalent or aggregative functional equivalents and derivatives thereofare useful as reagents in immunoassays or for affinity purificationprocedures. For example, a fragment of a mucin of the invention may bemade insoluble by covalent bonding to cyanogen bromide-activatedSepharose by methods known per se or adsorbed to polyolefin surfaces,either with or without glutaraldehyde cross-linking, for use in an assayor purification of anti-mucin antibodies or cell surface receptors.Fragments may also be labelled with a detectable group, e.g.,radioiodinated by the chloramine T procedure, covalently bound to rareearth chelates or conjugated to another fluorescent moiety for use ine.g. diagnostic assays.

Mutagenesis of a preferred predetermined fragment of a mucin epitope ofthe invention can be conducted by making amino acid insertions, usuallyon the order of about from 1 to 10 amino acid residues, preferably fromabout 1 to 5 amino acid residues, or deletions of from about from 1 to10 residues, such as from about 2 to 5 residues.

In one embodiment the fragment of the mucin epitope is synthesised byautomated synthesis. Any of the commercially available solid-phasetechniques may be employed, such as the Merrifield solid phase synthesismethod, in which amino acids are sequentially added to a growing aminoacid chain. (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963).

Equipment for automated synthesis of polypeptides is commerciallyavailable from suppliers such as Applied Biosystems, Inc. of FosterCity, Calif., and may generally be operated according to themanufacturer's instructions. Solid phase synthesis will enable theincorporation of desirable amino acid substitutions into any fragmentaccording to the present invention. It will be understood thatsubstitutions, deletions, insertions or any subcombination thereof maybe combined to arrive at a final sequence of a functional equivalent.Insertions shall be understood to include amino-terminal and/orcarboxyl-terminal fusions, e.g. with a hydrophobic or immunogenicprotein or a carrier such as any polypeptide or scaffold structurecapable as serving as a carrier.

Oligomers including dimers including homodimers and heterodimers offragments of mucin glycopeptides according to the invention are alsoprovided and fall under the scope of the invention. Mucin peptideequivalents and variants can be produced as homodimers or heterodimerswith other amino acid sequences or with native mucin sequences.Heterodimers include dimers containing immunoreactive mucin fragments aswell as mucin fragments that need not have or exert any biologicalactivity.

Mucin glycopeptide fragments according to the invention may besynthesised both in vitro and in vivo. Method for in vitro synthesis arewell known, and methods being suitable or suitably adaptable to thesynthesis in vivo are also described in the prior art. The GalNAc moietyof the glycans of the invention can be included in the peptide synthesisby the inclusion of GalNAc-S/T building blocks. When synthesized invivo, a host cell is transformed with vectors containing DNA encodingmucin fragments or a fragment thereof. A vector is defined as areplicable nucleic acid construct. Vectors are used to mediateexpression of mucin fragments. An expression vector is a replicable DNAconstruct in which a nucleic acid sequence encoding the predeterminedmucin fragment, or any functional equivalent thereof that can beexpressed in vivo, is operably linked to suitable control sequencescapable of effecting the expression of the fragment or equivalent in asuitable host. Such control sequences are well known in the art.

Cultures of cells derived from multicellular organisms representpreferred host cells. In principle, any higher eukaryotic cell cultureis workable, whether from vertebrate or invertebrate culture. Examplesof useful host cell lines are VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines.Preferred host cells are eukaryotic cells known to synthesize endogenousmucins. Cultures of such host cells may be isolated and used as a sourceof the fragment, or used in therapeutic methods of treatment, includingtherapeutic methods aimed at promoting or inhibiting a growth state, ordiagnostic methods carried out on the human or animal body.

Glycosylation

Glycosylation is the enzymatic process that links saccharides to produceglycans, attached to proteins, lipids, or other organic molecules. Thisenzymatic process produces one of the fundamental biopolymers found incells (along with DNA, RNA, and proteins). Glycosylation is a form ofco-translational and post-translational modification. Glycans serve avariety of structural and functional roles in membrane and secretedproteins. The majority of proteins synthesised in the rough ER undergoglycosylation. It is an enzyme-directed site-specific process, asopposed to the non-enzymatic chemical reaction of glycation.Glycosylation also occurs in the cytoplasm and nucleus, for example theO-GlcNAc modification. Five classes of glycan modification of peptidescan be produced: N-linked glycans attached to a nitrogen of asparagineor arginine side chains, O-linked glycans attached to the hydroxy oxygenof serine, threonine, tyrosine, hydroxylysine, or hydroxyproline sidechains, or to oxygens on lipids such as ceramide; phospho-glycans linkedthrough the phosphate of a phospho-serine; C-linked glycans, a rare formof glycosylation where a sugar is added to a carbon on a tryptophan sidechain, and glypiation which is the addition of a GPI anchor which linksproteins to lipids through glycan linkages.

The glycosylation of main interest in the present invention is O-linkedglycosylation. This type of glycosylation occurs at a late stage duringprotein processing, in the Golgi apparatus. This is the addition ofN-acetyl-galactosamine to serine or threonine residues by the enzymeUDP-N-acetyl-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase (EC 2.4.1.41), followed by othercarbohydrates (such as galactose and sialic acid). This process isimportant for certain types of proteins such as proteoglycans, whichinvolves the addition of glycosaminoglycan chains to an initiallyunglycosylated “proteoglycan core protein.” These additions are usuallyserine O-linked glycoproteins, which seem to have one of two mainfunctions. One function involves secretion to form components of theextracellular matrix, adhering one cell to another by interactionsbetween the large sugar complexes of proteoglycans. The other mainfunction is to act as a component of mucosal secretions, and it is thehigh concentration of carbohydrates that tends to give mucus itsconsistency. In addition, O-linked glycans are involved in directingcleavage of membrane proteins, intracellular sorting, secretion,protease resistance and in intracellular signalling.

In one embodiment of the present invention, at least one amino acidresidue, such as at least two amino acid residues, for example at leastthree amino acid residues, such as at least at least four amino acidresidues, for example at least five amino acid residues of the mucinpeptides defined herein above is/are glycosylated by an O-linked glycanselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

Each Serine (S) or Threonine (T) residue of the polypeptides selectedfrom the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3),PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO:11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ IDNO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21),PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ IDNO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24),PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25) andPMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGNGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFSSNPSRDSHTT(SEQ ID NO: 30) may be individually and optionally glycosylated by oneof the glycans selected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

Thus, in one embodiment of the present invention, one or more amino acidresidues of the mucin peptide is/are Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr)glycosylated.

In another embodiment of the present invention, one or more amino acidresidues of the mucin peptide is/are Tn (GalNAc-α-Ser/Thr) glycosylated.

In another embodiment of the present invention, one or more amino acidresidues of the mucin peptide is/are STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr) glycosylated.

In another embodiment of the present invention, one or more amino acidresidues of the mucin peptide is/are Core-2((Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) glycosylated.

In yet another embodiment of the present invention, one or more aminoacid residues of the mucin peptide is/are Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) glycosylated.

In yet another embodiment of the present invention, one or more aminoacid residues of the mucin peptide is/are ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr) glycosylated.

In one embodiment, the peptide used in the methods for detecting cancerdefined herein above, is selected from the group consisting of:VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) andPVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn)T(Tn)GHAT(Tn).

In one embodiment a glycopeptide selected from the group consisting ofVTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) andPVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn)T(Tn)GHAT(Tn)is used in a method of detecting IgG autoantibodies in a sample from asubject, wherein the presence of said autoantibodies to any of saidglycopeptides indicates colorectal cancer.

In one embodiment, the peptide of the present invention is selected fromthe group consisting of: VTSAPDT(Core3)RPAPGSTAPPAHG,VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG,VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG, VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC16Tn) andPVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn)T(Tn)GHAT(Tn),wherein Core-3 is GlcNAcβ1-3GalNAc-α-Ser/Thr, and wherein Tn isGalNAc-α-Ser/Thr, and wherein STn/sialyl-Tn isNeu5Acα2-6GalNAc-α-Ser/Thr, and wherein Core-2 isGalβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr and wherein Core-4 isGlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr, and wherein the glycosylation ison the serine (S) or Threonine (T) amino acid residue preceding theglycan denotation.

In one embodiment a glycopeptide selected from the group consisting ofVTSAPDT(Core3)RPAPGSTAPPAHG, VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG,VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG, VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC16Tn) andPVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn)T(Tn)GHAT(Tn),

is used in a method of detecting IgA autoantibodies in a sample from asubject, wherein the presence of said autoantibodies to any of saidglycopeptides indicates colorectal cancer.

Core-3 is an abbreviation for GlcNAcβ1-3GalNAc-α-Ser/Thr, and Tn is anabbreviation for GalNAc-α-Ser/Thr. STn or sialyl-Tn is an abbreviationfor Neu5Acα2-6GalNAc-α-Ser/Thr. Core-2 is an abbreviation forGalβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr. Core-4 is an abbreviation forGlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr. Glycosylation occurs on theserine (S) or Threonine (T) amino acid residue preceding the glycandenotation as defined herein above.

In one embodiment, the peptide of the invention is selected from thegroup consisting of PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*(MUC4-TR Tn) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*(MUC4 Tn), wherein the asterisk (*) indicates a potentialO-glycosylation site. Accordingly, each asterisk signifies that theamino acid residue preceding said asterisk can be glycosylated by anO-linked glycan selected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

Antibodies

Other aspects of the present invention are antibodies prepared using theglycopeptides defined herein above, methods for preparation of saidantibodies and use of said antibodies in therapy and diagnosis. Alsopart of the invention are antibodies capable of recognising theIO-glycosylated peptides of the present invention, wherein saidantibodies are produced by state-of-the art recombinant methods.

An antibody binds tightly to a particular target molecule, therebyeither inactivating it directly or marking it for destruction. Theantibody recognizes its target (antigen) with remarkable specificity andstrength dictated by the sum of many chemical forces, including hydrogenbonds, hydrophobic and van der Waal's forces, as well as ionicinteractions. In general, the more complex the target is chemically, themore immunogenic it will be. The antigenic determinant may encompassshort linear amino acid stretches or a more complicated,three-dimensional protein module.

Conceptually, antibodies directed against a target receptor may inhibitligand binding in two ways: competitive or allosteric. Competitiveinhibition involves the direct binding of the antibody to or near theligand binding site on the receptor, thereby displacing the ligand fromits receptor or sterically inhibiting the approach of the ligand to theligand binding site. Allosteric inhibition involves the binding of theantibody to a site on the receptor polypeptide that is distinct from theligand binding epitope. However, binding to this site will induce aconformational change in the overall structure of the receptor thatmakes it more difficult or even impossible for the ligand to bind to itscognate recognition site.

The antibody or functional equivalent thereof may be any antibody knownin the art, for example a polyclonal or a monoclonal antibody derivedfrom a mammal or a synthetic antibody, such as a single chain antibodyor hybrids comprising antibody fragments. Furthermore, the antibody maybe mixtures of monoclonal antibodies or artificial polyclonalantibodies. In addition functional equivalents of antibodies may beantibody fragments, in particular epitope binding fragments.Furthermore, antibodies or functional equivalent thereof may be a smallmolecule mimicking an antibody. Naturally occurring antibodies areimmunoglobulin molecules consisting of heavy and light chains. Inpreferred embodiments of the invention, the antibody is a monoclonalantibody.

Monoclonal antibodies (Mab's) are antibodies, wherein every antibodymolecule are similar and thus recognises the same epitope. Monoclonalantibodies are in general produced by a hybridoma cell line. Methods ofmaking monoclonal antibodies and antibody-synthesizing hybridoma cellsare well known to those skilled in the art. Antibody producinghybridomas may for example be prepared by fusion of an antibodyproducing B lymphocyte with an immortalized B-lymphocyte cell line.Monoclonal antibodies according to the present invention may for examplebe prepared as described in Antibodies: A Laboratory Manual, By EdHarlow and David Lane, Cold Spring Harbor Laboratory Press, 1988. Saidmonoclonal antibodies may be derived from any suitable mammalianspecies, however frequently the monoclonal antibodies will be rodentantibodies for example murine or rat monoclonal antibodies. It ispreferred that the antibodies according to the present invention aremonoclonal antibodies or derived from monoclonal antibodies.

Polyclonal antibodies is a mixture of antibody molecules recognising aspecific given antigen, hence polyclonal antibodies may recognisedifferent epitopes within said antigen. In general polyclonal antibodiesare purified from serum of a mammal, which previously has been immunizedwith the antigen. Polyclonal antibodies may for example be prepared byany of the methods described in Antibodies: A Laboratory Manual, By EdHarlow and David Lane, Cold Spring Harbor Laboratory Press, 1988.Polyclonal antibodies may be derived from any suitable mammalianspecies, for example from mice, rats, rabbits, donkeys, goats, sheeps,cows or camels. The antibody is preferably not derived from anon-mammalian species, i.e. the antibody is for example preferably not achicken antibody. The antibody may also for example be an artificialpolyclonal antibody as for example described in U.S. Pat. Nos. 5,789,208or 6,335,163, both patent specifications are hereby incorporated byreference into the application in their entirety.

The antibodies according to the present invention may also berecombinant antibodies. Recombinant antibodies are antibodies orfragments thereof or functional equivalents thereof produced usingrecombinant technology. For example recombinant antibodies may beproduced using a synthetic library or by phage display. Recombinantantibodies may be produced according to any conventional method forexample the methods outlined in “Recombinant Antibodies”, FrankBreitling, Stefan Dübel, Jossey-Bass, September 1999.

The antibodies according to the present invention may also be bispecificantibodies, i.e. antibodies specifically recognising two differentepitopes. Bispecific antibodies may in general be prepared starting frommonoclonal antibodies, or from recombinant antibodies, for example byfusing two hybridoma's in order to combine their specificity, byChemical crosslinking or using recombinant technologies. Antibodiesaccording to the present invention may also be tri-specific antibodies.

Functional equivalents of antibodies may in one preferred embodiment bea fragment of an antibody, preferably an antigen binding fragment or avariable region. Examples of antibody fragments useful with the presentinvention include Fab, Fab′, F(ab′)₂ and Fv fragments. Papain digestionof antibodies produces two identical antigen binding fragments, calledthe Fab fragment, each with a single antigen binding site, and aresidual “Fc” fragment, so-called for its ability to crystallizereadily. Pepsin treatment yields an F(ab′)₂ fragment that has twoantigen binding fragments which are capable of cross-linking antigen,and a residual other fragment (which is termed pFc′). Additionalfragments can include diabodies, linear antibodies, single-chainantibody molecules, and multispecific antibodies formed from antibodyfragments. As used herein, “functional fragment” with respect toantibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

Preferred antibody fragments retain some or essential all the ability ofan antibody to selectively binding with its antigen or receptor. Somepreferred fragments are defined as follows:

-   -   (1) Fab is the fragment that contains a monovalent        antigen-binding fragment of an antibody molecule. A Fab fragment        can be produced by digestion of whole antibody with the enzyme        papain to yield an intact light chain and a portion of one heavy        chain.    -   (2) Fab′ is the fragment of an antibody molecule and can be        obtained by treating whole antibody with pepsin, followed by        reduction, to yield an intact light chain and a portion of the        heavy chain. Two Fab′ fragments are obtained per antibody        molecule. Fab′ fragments differ from Fab fragments by the        addition of a few residues at the carboxyl terminus of the heavy        chain CH1 domain including one or more cysteines from the        antibody hinge region.    -   (3) (Fab′)₂ is the fragment of an antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction. F(ab′)₂ is a dimer of two Fab′ fragments        held together by two disulfide bonds.    -   (4) Fv is the minimum antibody fragment that contains a complete        antigen recognition and binding site. This region consists of a        dimer of one heavy and one light chain variable domain in a        tight, non-covalent association (V_(H)-V_(L) dimer). It is in        this configuration that the three CDRs of each variable domain        interact to define an antigen binding site on the surface of the        V_(H)-V_(L) dimer. Collectively, the six CDRs confer antigen        binding specificity to the antibody. However, even a single        variable domain (or half of an Fv comprising only three CDRs        specific for an antigen) has the ability to recognize and bind        antigen, although at a lower affinity than the entire binding        site.

In one embodiment of the present invention the antibody is a singlechain antibody (“SCA”), defined as a genetically engineered moleculecontaining the variable region of the light chain, the variable regionof the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Such single chain antibodiesare also refered to as “single-chain Fv” or “scFv” antibody fragments.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains that enables the scFv to form the desiredstructure for antigen binding.

In another embodiment of the present invention the functional equivalentof an antibody is a small molecule mimicking an antibody. Such moleculesmay be a non-immunoglobulin binding members. Thus the epitopepolypeptide of the present invention binding may be derived from anaturally occurring protein or polypeptide; said protein or polypeptidemay for example be designed de novo, or may be selected from a library.The binding member may be a single moiety, e.g., a polypeptide orprotein domain, or it may include two or more moieties, e.g., a pair ofpolypeptides such as a pair polypeptides. The binding polypeptide mayfor example, but not exclusively, be a lipocalin, a single chain MHCmolecule, an Anticalin™ (Pieris), an Affibody™, or a Trinectin™(Phylos), Nanobodies (Ablynx). The binding member may be selected ordesigned by recombinant methods known by people well known in the art.

Human monoclonal antibodies of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein, Nature 256:495 (1975). Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed, e.g., viral oroncogenic transformation of B-lymphocytes or phage display techniquesusing libraries of human antibody genes.

Immunizations

To generate fully human monoclonal antibodies to the epitopes ofinterest to the present invention, transgenic or transchromosomal micecontaining human immunoglobulin genes can be immunized with an enrichedpreparation of the antigen and/or cells expressing the epitopes of thereceptor targets of the present invention, as described, for example, byLonberg et al. (1994), supra; Fishwild et al. (1996), supra, and WO98/24884. Alternatively, mice can be immunized with DNA encoding theCaOU-1 epitope. Preferably, the mice will be 6-16 weeks of age upon thefirst infusion.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(i.p.) or subcutaneously (s.c.) with antigen expressing cells incomplete Freund's adjuvant, followed by every other week i.p.immunizations (up to a total of 10) with the antigen expressing cells inPBS. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by FACS analysis, and mice withsufficient titers of anti-antigen human immunoglobulin can be used forfusions. Mice can be boosted intravenously with antigen expressing cellsfor example 4 and 3 days before sacrifice and removal of the spleen.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C.et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such frameworksequences can be obtained from public DNA databases that includegermline antibody gene sequences. These germline sequences will differfrom mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody whichcontains mutations throughout the variable gene but typically clusteredin the CDRs. For example, somatic mutations are relatively infrequent inthe amino terminal portion of framework region 1 and in thecarboxy-terminal portion of framework region 4. For this reason, it isnot necessary to obtain the entire DNA sequence of a particular antibodyin order to recreate an intact recombinant antibody having bindingproperties similar to those of the original antibody (see WO 99/45962).Partial heavy and light chain sequence spanning the CDR regions istypically sufficient for this purpose. The partial sequence is used todetermine which germline variable and joining gene segments contributedto the recombined antibody variable genes. The germline sequence is thenused to fill in missing portions of the variable regions. Heavy andlight chain leader sequences are cleaved during protein maturation anddo not contribute to the properties of the final antibody. To addmissing sequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone.

This process has certain advantages such as elimination or inclusion orparticular restriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts fromhybridomas are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266:19867-19870); and HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotides approximately at the midpoint of thecorresponding non-coding oligonucleotide. Thus, for each chain, theoligonucleotides can be assembled into overlapping double stranded setsthat span segments of 150-400 nucleotides. The pools are then used astemplates to produce PCR amplification products of 150-400 nucleotides.Typically, a single variable region oligonucleotide set will be brokendown into two pools which are separately amplified to generate twooverlapping PCR products. These overlapping products are then combinedby PCR amplification to form the complete variable region. It may alsobe desirable to include an overlapping fragment of the heavy or lightchain constant region (including the BbsI site of the kappa light chain,or the AgeI site of the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader, translation initiation, constantregion, 3′ untranslated, polyadenylation, and transcription termination,sequences to form expression vector constructs. The heavy and lightchain expression constructs can be combined into a single vector,co-transfected, serially transfected, or separately transfected intohost cells which are then fused to form a host cell expressing bothchains.

Monovalent Antibodies

The monospecific binding polypeptide may be monovalent, i.e. having onlyone binding domain.

For a monovalent antibody, the immunoglobulin constant domain amino acidresidue sequences comprise the structural portions of an antibodymolecule known in the art as CH1, CH2, CH3 and CH4. Preferred are thosebinding polypeptides which are known in the art as C_(L). PreferrredC_(L) polypeptides are selected from the group consisting of C_(kappa)and C_(lambda).

Furthermore, insofar as the constant domain can be either a heavy orlight chain constant domain (C_(H) or C_(L), respectively), a variety ofmonovalent binding polypeptide compositions are contemplated by thepresent invention. For example, light chain constant domains are capableof disulfide bridging to either another light chain constant domain, orto a heavy chain constant domain. In contrast, a heavy chain constantdomain can form two independent disulfide bridges, allowing for thepossibility of bridging to both another heavy chain and to a lightchain, or to form polymers of heavy chains.

Thus, in another embodiment, the invention contemplates an isolatedmonovalent binding polypeptide wherein the constant chain domain C has acysteine residue capable of forming at least one disulfide bridge, andwhere at least two monovalent polypeptides are covalently linked by saiddisulfide bridge.

In preferred embodiments, the constant chain domain C can be eitherC_(L) or C_(H). Where C is C_(L), the C_(L) polypeptide is preferablyselected from the group consisting of C_(kappa) and C_(lambda).

In another embodiment, the invention contemplates a binding polypeptidecomposition comprising a monovalent polypeptide as above except where Cis C_(L) having a cysteine residue capable of forming a disulfidebridge, such that the composition contains two monovalent polypeptidescovalently linked by said disulfide bridge.

Multispecificity, Including Bispecificity

In a preferred embodiment the present invention relates to multispecificbinding polypeptides, which have affinity for and are capable of bindingat least two different entities. Multispecific binding polypeptides caninclude bispecific binding polypeptides.

In one embodiment the multispecific molecule is a bispecific antibody(BsAb), which carries at least two different binding domains, wherepreferably at least one of which is of antibody origin.

A bispecific molecule of the invention can also be a single chainbispecific molecule, such as a single chain bispecific antibody, asingle chain bispecific molecule comprising one single chain antibodyand a binding domain, or a single chain bispecific molecule comprisingtwo binding domains. Multispecific molecules can also be single chainmolecules or may comprise at least two single chain molecules.

The multispecific, including bispecific, antibodies may be produced byany suitable manner known to the person skilled in the art.

The traditional approach to generate bispecific whole antibodies was tofuse two hybridoma cell lines each producing an antibody having thedesired specificity. Because of the random association of immunoglobulinheavy and light chains, these hybrid hybridomas produce a mixture of upto 10 different heavy and light chain combinations, only one of which isthe bispecific antibody. Therefore, these bispecific antibodies have tobe purified with cumbersome procedures, which considerably decrease theyield of the desired product.

Alternative approaches include in vitro linking of two antigenspecificities by chemical cross-linking of cysteine residues either inthe hinge or via a genetically introduced C-terminal Cys as describedabove. An improvement of such in vitro assembly was achieved by usingrecombinant fusions of Fab's with peptides that promote formation ofheterodimers. However, the yield of bispecific product in these methodsis far less than 100%.

A more efficient approach to produce bivalent or bispecific antibodyfragments, not involving in vitro chemical assembly steps, was describedby Holliger et al. (1993). This approach takes advantage of theobservation that scFv's secreted from bacteria are often present as bothmonomers and dimers. This observation suggested that the V_(H) and V_(L)of different chains could pair, thus forming dimers and largercomplexes. The dimeric antibody fragments, also named “diabodies” byHollinger et al., are in fact small bivalent antibody fragments thatassembled in vivo. By linking the V_(H) and V_(L) of two differentantibodies 1 and 2, to form “cross-over” chains V_(H) 1V_(L) 2 and V_(H)2 -V_(L) 1, the dimerisation process was shown to reassemble bothantigen-binding sites. The affinity of the two binding sites was shownto be equal to the starting scFv's, or even to be 10-fold increased whenthe polypeptide linker covalently linking V_(H) and V_(L) was removed,thus generating two proteins each consisting of a V_(H) directly andcovalently linked to a V_(L) not pairing with the V_(H). This strategyof producing bispecific antibody fragments was also described in severalpatent applications. Patent application WO 94/09131 (SCOTGEN LTD;priority date Oct. 15, 1992) relates to a bispecific binding protein inwhich the binding domains are derived from both a V_(H) and a V_(L)region either present at two chains or linked in an scFv, whereas otherfused antibody domains, e.g. C-terminal constant domains, are used tostabilise the dimeric constructs. Patent application WO 94/13804(CAMBRIDGE ANTIBODY TECHNOLOGY/MEDICAL RESEARCH COUNCIL; first prioritydate Dec. 4, 1992) relates to a polypeptide containing a V_(H) and aV_(L) which are incapable of associating with each other, whereby theV-domains can be connected with or without a linker.

Mallender and Voss, 1994 (also described in patent application WO94/13806; DOW CHEMICAL CO; priority date Dec. 11, 1992) reported the invivo production of a single-chain bispecific antibody fragment in E.coli. The bispecificity of the bivalent protein was based on twopreviously produced monovalent scFv molecules possessing distinctspecificities, being linked together at the genetic level by a flexiblepolypeptide linker. Traditionally, whenever single-chain antibodyfragments are referred to, a single molecule consisting of one heavychain linked to one (corresponding) light chain in the presence orabsence of a polypeptide linker is implicated. When making bivalent orbispecific antibody fragments through the “diabody” approach (Holligeret al., (1993) and patent application WO 94/09131) or by the “doublescFv” approach (Mallender and Voss, 1994 and patent application WO94/13806), again the V_(H) is linked to a (the corresponding) V_(L).

The multispecific molecules described above can be made by a number ofmethods. For example, all specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the multi-specific molecule is a mAb X mAb,mAb X Fab, Fab X F(ab′)₂ or ligand X Fab fusion protein. Various othermethods for preparing bi- or multivalent antibodies are described forexample described in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175;5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

By using a bispecific or multispecific binding polypeptide according tothe invention the invention offers several advantages as compared tomonospecific/monovalent binding polypeptides.

It may be preferred that the at least one other binding domain iscapable of binding an immunoactive cell, such as a leucocyte, amacrophage, a lymphocyte, a basophilic cell, and/or an eosinophiliccell, in order to increase the effect of the binding polypeptide in atherapeutic method. This may be accomplished by establishing that the atleast one other binding domain is capable of specifically binding amammalian protein, such as a human protein, such as a protein selectedfrom any of the cluster differentiation proteins (CD), in particularCD64 and/or CD89. A method for producing bispecific antibodies havingCD64 specificity is described in U.S. Pat. No. 6,071,517 to Medarex,Inc. The production and characterization of these preferred monoclonalantibodies are described by Fanger et al. in WO 88/00052 and in U.S.Pat. No. 4,954,617.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific or multispecific molecules of theinvention are murine, chimeric and humanized monoclonal antibodies. Suchmurine, chimeric and humanized monoclonal antibodies can be prepared bymethods known in the art.

Bispecific and multispecific molecules of the present invention can bemade using chemical techniques (see e.g., D. M. Kranz et al. (1981)Proc. Natl. Acad. Sci. USA 78:5807), “polydoma” techniques (see U.S.Pat. No. 4,474,893), or recombinant DNA techniques.

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific and multispecific molecule is amAb x mAb, mAb x Fab, Fab x F(ab′)_(h2) or ligand x Fab fusion protein.A bispecific and multispecific molecule of the invention, e.g., abispecific molecule can be a single chain molecule, such as a singlechain bispecific antibody, a single chain bispecific molecule comprisingone single chain antibody and a binding determinant, or a single chainbispecific molecule comprising two binding determinants. Bispecific andmultispecific molecules can also be single chain molecules or maycomprise at least two single chain molecules. Methods for preparing bi-and multispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific and multispecific molecules to their specifictargets can be confirmed by enzyme-linked immunosorbent assay (ELISA), aradioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growthinhibition), or a Western Blot Assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986).The radioactive isotope can be detected by such means as the use of a γcounter or a scintillation counter or by autoradiography.

Humanised Antibody Framework

It is not always desirable to use non-human antibodies for humantherapy, since the non-human “foreign” epitopes may elicit immuneresponse in the individual to be treated. To eliminate or minimize theproblems associated with non-human antibodies, it is desirable toengineer chimeric antibody derivatives, i.e., “humanized” antibodymolecules that combine the non-human Fab variable region bindingdeterminants with a human constant region (Fc). Such antibodies arecharacterized by equivalent antigen specificity and affinity of themonoclonal and polyclonal antibodies described above, and are lessimmunogenic when administered to humans, and therefore more likely to betolerated by the individual to be treated.

Humanised antibodies are in general chimeric antibodies comprisingregions derived from a human antibody and regions derived from anon-human antibody, such as a rodent antibody. Humanisation (also calledReshaping or CDR-grafting) is a well-established technique for reducingthe immunogenicity of monoclonal antibodies (mAbs) from xenogeneicsources (commonly rodent), increasing the homology to a humanimmunoglobulin, and for improving their activation of the human immunesystem. Thus, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some framework residues aresubstituted by residues from analogous sites in rodent antibodies.

It is further important that humanized antibodies retain high affinityfor the antigen and other favourable biological properties. To achievethis goal, according to a preferred method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of certain residues in the functioning ofthe candidate immunoglobulin sequence, i.e., the analysis of residuesthat influence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), ismaximized, although it is the CDR residues that directly and mostsubstantially influence antigen binding.

One method for humanising MAbs related to production of chimericantibodies in which an antigen binding site comprising the completevariable domains of one antibody are fused to constant domains derivedfrom a second antibody, preferably a human antibody. Methods forcarrying out such chimerisation procedures are for example described inEP-A-0 120 694 (Celltech Limited), EP-A-0 125 023 (Genentech Inc.),EP-A-0 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (StanfordUniversity) and EP-A-0 194 276 (Celltech Limited). A more complex formof humanisation of an antibody involves the re-design of the variableregion domain so that the amino acids constituting the non-humanantibody binding site are integrated into the framework of a humanantibody variable region (Jones et al., 1986).

The humanized antibody of the present invention may be made by anymethod capable of replacing at least a portion of a CDR of a humanantibody with a CDR derived from a non-human antibody. Winter describesa method which may be used to prepare the humanized antibodies of thepresent invention (UK Patent Application GB 2188638A, filed on Mar. 26,1987), the contents of which is expressly incorporated by reference. Thehuman CDRs may be replaced with non-human CDRs using oligonucleotidesite-directed mutagenesis as described in the examples below.

As an example the humanized antibody of the present invention may bemade as described in the brief explanation below. The humanizedantibodies of the present invention may be produced by the followingprocess:

-   (a) constructing, by conventional techniques, an expression vector    containing an operon with a DNA sequence encoding an antibody heavy    chain in which the CDRs and such minimal portions of the variable    domain framework region that are required to retain antibody binding    specificity are derived from a non-human immunoglobulin, and the    remaining parts of the antibody chain are derived from a human    immunoglobulin, thereby producing the vector of the invention;-   (b) constructing, by conventional techniques, an expression vector    containing an operon with a DNA sequence encoding a complementary    antibody light chain in which the CDRs and such minimal portions of    the variable domain framework region that are required to retain    donor antibody binding specificity are derived from a non-human    immunoglobulin, and the remaining parts of the antibody chain are    derived from a human immunoglobulin, thereby producing the vector of    the invention;-   (c) transfecting the expression vectors into a host cell by    conventional techniques to produce the transfected host cell of the    invention; and-   (d) culturing the transfected cell by conventional techniques to    produce the humanised antibody of the invention.

The host cell may be cotransfected with the two vectors of theinvention, the first vector containing an operon encoding a light chainderived polypeptide and the second vector containing an operon encodinga heavy chain derived polypeptide. The two vectors contain differentselectable markers, but otherwise, apart from the antibody heavy andlight chain coding sequences, are preferably identical, to ensure, asfar as possible, equal expression of the heavy and light chainpolypeptides. Alternatively, a single vector may be used, the vectorincluding the sequences encoding both the light and the heavy chainpolypeptides. The coding sequences for the light and heavy chains maycomprise cDNA or genomic DNA or both.

The host cell used to express the altered antibody of the invention maybe either a bacterial cell such as E. coli, or a eukaryotic cell. Inparticular a mammalian cell of a well defined type for this purpose,such as a myeloma cell or a Chinese hamster ovary cell may be used.

The general methods by which the vectors of the invention may beconstructed, transfection methods required to produce the host cell ofthe invention and culture methods required to produce the antibody ofthe invention from such host cells are all conventional techniques.Likewise, once produced, the humanized antibodies of the invention maybe purified according to standard procedures as described below.

Antigenic epitope(s) such as the O-glycosylated peptides of theinvention may be administered to a mammal in an amount sufficient tostimulate an immunological response against the antigenic epitope(s).The antigenic epitope(s) may be combined in a therapeutic compositionand administered in several doses over a period of time that optimizesthe immunological response of the mammal. Such an immunological responsecan be detected and monitored by observing whether antibodies directedagainst the epitopes of the invention are present in the bloodstream ofthe mammal.

Such antibodies can be used alone or conjugated to, or combined with,therapeutically useful agents. Antibodies can be administered to mammalssuffering from any cancer that displays the cancer-associatedepitope(s). Such administration can provide both therapeutic treatment,and prophylactic or preventative measures. For example, therapeuticmethods can be used to determine the spread of a cancer and lead to itsremission.

Antibodies of the invention can be used for passive immunization ofpatient, i.e. administering the antibodies or as antibody fragments suchas Fab fragments in isolated form to the patient. Furthermore,medicaments such as toxins or chemotherapeutic agents can be conjugatedto the antibodies of the invention by methods known to those skilled inthe art. All of the above antibodies can subsequent to administrationtarget cancer cells specifically, based on the knowledge that thepattern of multiple and aberrantly O-glycosylated mucins on cancer cellsdistinguishes them from healthy cells.

Therapeutically useful agents which may be conjugated to the antibodiesof the invention include but is not limited to the group comprisingadrimycin, aminoglutethimide, aminopterin, azathioprine, bleomycinsulfate, bulsulfan, carboplatin, carminomycin, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclosporine, cytarabidine, cytosinearabinoside, cytoxin dacarbazine, dactinomycin, daunomycin,daunorubicin, doxorubicin, esperamicins, etoposide, fluorouracil,ifosfamide, interferon-α, lomustine, melphalan, mercaptopurine,methotrexate, mitomycin C, mitotane, mitoxantrone, procarbazine HCl,taxol, taxotere (docetaxel), teniposide, thioguanine, thiotepa,vinblastine sulfate, vincristine sulfate and vinorelbine. Additionalagents include those disclosed in Chapter 52, Antineoplastic Agents(Paul Calabresi and Bruce A. Chabner), and the introduction thereto, pp.1202-1263, of Goodman and Gilman's “The Pharmacological Basis ofTherapeutics”, Eighth Edition, 1990, McGraw-Hill, Inc. (HealthProfessions Division). Toxins can be proteins such as, for example,pokeweed anti-viral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin, or Pseudomonas exotoxin. Toxinmoieties can also be high energy-emitting radionuclides such ascobalt-60, I-131, I-125, Y-90 and Re-186, and enzymatically activetoxins of bacterial, fungal, plant or animal origin, or fragmentsthereof.

Chemotherapeutic agents can be used to reduce the growth or spread ofcancer cells and tumors that express the tumor associated epitope of theinvention. Animals that can be treated by the chemotherapeutic agents ofthe invention include humans, non-human primates, cows, horses, pigs,sheep, goats, dogs, cats, rodents and the like. In all embodiments humantumor antigens and human subjects are preferred.

Species-dependent antibodies can be used in therapeutic methods. Such aspecies-dependent antibody has constant regions that are substantiallynon-immunologically reactive with the chosen species. Suchspecies-dependent antibody is particularly useful for therapy because itgives rise to substantially no immunological reactions. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is mammalian, and morepreferably is a humanized or human antibody.

The present inventors have found glycopeptide epitopes associated withcolorectal cancer and antibodies useful in detecting said glycopeptides.

In another aspect, the present invention relates to a method fordetecting colorectal cancer, said method comprising

(i) contacting a sample from said host organism with one or moreO-glycosylated mucin peptides, wherein said peptide comprises, or saidpeptides comprise, at least 5 consecutive amino acid residues of amucin, or a fragment or variant thereof, wherein said variant is atleast 70% identical, such as at least 75% identical to, e.g. at least80% identical to, such as at least 85% identical to, e.g. at least 90%identical to, such as at least 95% identical to, e.g. at least 98%identical to, such as at least 99% identical to said at least 5consecutive amino acid residues of said mucin, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe host organism.

The present inventors have raised several antibodies specific fordifferent glycosylated parts of the mucin proteins. Accordingly, in oneaspect the present invention relates to a method for detecting cancer,said method comprising

(i) contacting a sample with one or more antibodies capable ofrecognising one or more O-glycosylated mucin peptides, wherein saidpeptide comprises at least 5 consecutive amino acid residues of a mucin,or a fragment or variant thereof, wherein said variant is at least 70%identical, such as at least 75% identical to, e.g. at least 80%identical to, such as at least 85% identical to, e.g. at least 90%identical to, such as at least 95% identical to, e.g. at least 98%identical to, such as at least 99% identical to said at least 5consecutive amino acid residues of said mucin, and

(ii) removing unbound sample and/or unbound antibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In one embodiment said O-glycosylated mucin peptide is selected from thegroup consisting of

a) MUC4 Tn selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

b) a MUC4 non glycosylated mucin peptide selected from the groupconsisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ IDNO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a Tnglycosylated mucin peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), whereinthe asterisk (*) indicates a potential O-glycosylation site, wherein theoptional glycan is Tn (GalNAc-α-Ser/Thr), or

c) a Tn glycosylated MUC4 peptide selected from the group consisting ofPMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

d) an all-Tn MUC4 peptide selected from the group consisting ofPVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) andPMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQIDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T*(SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylationsite, wherein the glycan is Tn (GalNAc-α-Ser/Thr), or

e) a recombinant MUC4 Tn having the sequencePMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQID NO: 30), wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is Tn(GalNAc-α-Ser/Thr), or

f) a MUC1Tn/STn/Core3 glycosylated or MUC4 glycosylated mucin peptideselected from the group consisting ofVT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG,VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG,PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS*(SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23),

and

PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQID NO: 30),

wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), or

g) a MUC1 STn and a MUC4 selected from the group consisting ofVT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG,PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) andPVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), andwherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In one aspect the present invention relates to a method for detecting agastrointestinal disease in a host organism wherein said disease ischaracterised in that O-glycosylated mucin peptides are shed from thediseased host and secreted in the gastrointestinal tract of the sampleorganism suffering from the disease, said method comprising

(i) contacting a sample from said host organism with one or moreantibodies capable of recognising said O-glycosylated mucin peptides,and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to the antibodies of step (i) areindicative of disease or disorder in the host organism.

In another aspect the present invention relates to a method fordetecting a gastrointestinal disease in a host organism wherein saiddisease is characterised in that O-glycosylated mucin peptides are shedfrom the diseased cells of the host into an extracellular volume, suchas secreted into, the lumen of the bladder, milk ducts of the breast,lumen of the uterus, the vagina, into pancreatic fluid, into ascitesfluid, onto bronchiolar surface of the lung, ductal surfaces of theprostate, lumen of the seminiferous tubules, the oesophagus or thegastrointestinal tract of the sample organism suffering from thedisease, said method comprising

(i) contacting a sample from said host organism with one or moreantibodies capable of recognising said O-glycosylated mucin peptides,and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to the antibodies of step (i) areindicative of disease or disorder in the host organism.

In one embodiment the disease detected by the above method is cancerwherein the cancer is selected from the group consisting of colorectalcancer, breast cancer, oral cancer, gastric cancer, esophageal cancer,pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer,renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer,basal cell cancer, squamous cell cancer, malignant melanoma, bladdercancer, endometrial cancer and cervix cancer.

In one embodiment the disease detected by the above method is colorectalcancer.

In one embodiment of the above method, the antibody the presentinvention is capable of recognising at least one O-glycosylated peptide,said peptide comprising at least 5 consecutive amino acid residues of amucin selected from the group consisting of MUC1 Variant CT58, MUC1Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1Variant ZD,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8,MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18,MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variant thereof,wherein said variant is at least 70% identical to said at least 5consecutive amino acid residues of said mucin.

In one embodiment of the present invention the at least 5 consecutiveamino acid residues of a mucin as defined herein above are from a mucinselected from the group consisting of MUC1 Variant CT58, MUC1 VariantCT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1 VariantZD,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9,MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19,MUC20, MUC21 and MUC-HEG.

In one embodiment the antibody is capable of recognising at least oneO-glycosylated peptide variant wherein said variant is at least 70%identical a peptide selected from the group consisting ofPMTDTKTVTTPGSSFTA (SEQ ID NO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4),SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO:6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO:8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO:10), TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO:12), EASGQTQTSEPASSGSRTT (SEQ ID NO: 13), PASSGSRTTSAGTATPSSS (SEQ IDNO: 14), TATPSSSGASGTTPSGSEGI (SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17),PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ IDNO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23),VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), andPTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25).

In another embodiment the antibody is capable of recognising anO-glycosylated mucin peptide selected from the group consisting ofVTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4),S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A(SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7),APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P(SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10),T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T*(SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQID NO: 30),

wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In one aspect the invention relates to a method for detecting colorectalcancer, said method comprising

(i) contacting a sample with a polyclonal antibody serum wherein saidpolyclonal antibody is capable of recognising at least two differentO-glycosylated mucin peptides, wherein said peptides comprises at least5 consecutive amino acid residues selected from the group consisting ofMUC1 Variant CT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X,MUC1 Variant Y, MUC1 Variant ZD,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B,MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16,MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variantthereof, wherein said variant is at least 70% identical to said at least5 consecutive amino acid residues of said mucin, and

(ii) removing unbound sample and/or unbound antibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In one embodiment the at least two different O-glycosylated mucinpeptides as defined herein above are a first O-glycosylated mucinpeptide selected from the group consisting of PMTDTKTVTTPGSSFTA (SEQ IDNO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ IDNO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ IDNO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ IDNO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ IDNO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQID NO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI(SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT(SEQ ID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21),PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ IDNO: 23) and PMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGNGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETT RFSSNPS (SEQID NO: 30) wherein at least one serine and/or threonine residue isoptionally O-glycosylated and wherein the optional glycan is selectedfrom the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and a second O-glycosylated mucinpeptide selected from the group consisting ofVTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) respectively, and

(ii) removing unbound sample, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein antibodies bound to said peptides indicate cancer inthe sample host.

In one aspect the invention relates to a method for detecting cancer,said method comprising

(i) contacting a sample with at least one first and at least one secondantibody, wherein said first antibody is capable of recognising a firstmucin peptide selected from the group consisting of the O-glycosylatedMUC1 peptides VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3),VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3),VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3),VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn),VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn),VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn),VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn),VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn),

and wherein said second antibody is capable of recognising another mucinpeptide selected from: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQIDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT*(SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15),S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) andGET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17),PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18),LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19),LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20),PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21),S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22),PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*TTS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ IDNO: 30), wherein the asterisk (*) indicates a potential O-glycosylationsite, wherein the optional glycan is independently selected from thegroup consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3GalβGalNAc-Ser/Thr), and

(ii) removing unbound sample and/or unbound antibody), and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In another aspect the invention relates to a method for detectingcancer, said method comprising

(i) contacting a sample with at least one antibody, wherein saidantibody is capable of recognising a glycosylated mucin peptide, whereinsaid peptide comprises at least 5 consecutive amino acid residues of amucin selected from the group consisting of MUC4 (SEQ ID NO: 1) and/orMUC1 (SEQ ID NO: 2), or a fragment or variant thereof, wherein saidvariant is at least 70% identical to said at least 5 consecutive aminoacid residues of said mucin selected from the group consisting of MUC4(SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), and

(ii) removing unbound sample and/or unbound antibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In one aspect the invention relates to a method for detecting colorectalcancer, said method comprising

(i) contacting a sample with at least one antibody, wherein saidantibody is capable of recognising a glycosylated mucin peptide selectedfrom the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3),PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO:11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ IDNO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18),LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19),LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20),PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21),PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ IDNO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), andPTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25) or a fragment of saidpeptides, or variants of said peptides in which variants any amino acidhas been changed to a different amino acid, provided that no more than 5of the amino acid residues in the sequence are so changed, and

(ii) removing unbound sample and/or unbound antibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

In another aspect the invention relates to a method for detectingcolorectal cancer, said method comprising

(i) contacting a sample with at least two different antibodies, whereinsaid antibodies are capable of recognising two different glycosylatedmucin peptides, wherein said peptide comprises at least 5 consecutiveamino acid residues of a mucin selected from the group consisting ofMUC4 (SEQ ID NO:1), MUC1 (SEQ ID NO: 2), MUC2 (SEQ ID NO: 26), MUC5AC(SEQ ID NO: 27), MUC6 (SEQ ID NO: 28), and MUC7 (SEQ ID NO: 29) or anaturally occurring fragment or variant of said mucin, wherein saidvariant is at least 70% identical to said at least 5 consecutive aminoacid residues of said mucin, and

(ii) removing unbound sample and/or unbound antibody, and

(iii) qualitatively and/or quantitatively characterise the boundmaterial, wherein peptides bound to said antibodies indicate cancer inthe sample host.

The antibodies of the present invention have been raised againstdifferent parts of glycosylated mucin polypeptides, Thus in one mainaspect, the present invention relates to a an antibody, in particular amonoclonal antibody as defined in the claims 1 to 5. The invention alsorelates to antigen binding fragment of said antibody, wherein saidantibody or the antigen binding fragment of said antibody is capable ofspecifically recognising a mucin glycopeptide as defined in any of theclaims.

In one embodiment the antibody is selected from the group consisting ofIgA, IgG, IgD, IgE and IgM antibodies.

In a further embodiment the IgA antibody is an IgA1 or an IgA2 antibody.

In a further embodiment the IgG antibody is selected from the groupconsisting of IgG1, IgG2, IgG3 and IgG4 antibodies.

In a further embodiment the IgG antibody is selected from the groupconsisting of mouse IgG1, mouse IgG2A, mouse IgG2B and mouse IgG3antibodies.

In a further embodiment the IgG antibody is selected from the groupconsisting of human IgG1, human IgG2, human IgG3 and human IgG4antibodies.

In a further embodiment the IgG antibody is selected from the groupconsisting of rabbit IgG1, rabbit IgG2A, rabbit IgG2B and rabbit IgG3antibodies.

In a further embodiment the IgG antibody is selected from the groupconsisting of goat IgG1, goat IgG2A, goat IgG2B and goat IgG3antibodies.

In one embodiment the antibodies of the invention is raised using anyother mammal suitable, as known to the person skilled in the art, forthe purpose of raising antibodies.

In one embodiment the antibody of the present invention is selected fromthe group consisting of MAb 5C10, MAb 3C9, MAb 4D9, MAb 6C11 and MAb6E3.

In one embodiment the antibody is MAb 4D9 produced by the cell line 4D9deposited under the Budapest Treaty with the European Collection ofAuthenticated Cell Cultures (ECACC) on Dec. 1, 2009, and which was givenaccession number 09120102. The address of the depository is PublicHealth England Culture Collections, Porton Down, Salisbury SP4 OJG,United Kingdom.

In one embodiment the antibody is MAb 5C10 produced by the cell line5C10 deposited under the Budapest Treaty with the ECACC on Dec. 1, 2009,and which was given accession number 09120101.

In one embodiment the antibody is MAb 6E3 produced by the cell line 6E3deposited under the Budapest Treaty with the ECACC on Dec. 1, 2009, andwhich was given accession number 09120103.

In one embodiment the antibody is MAb 3C9.

In one embodiment the antibody is MAb 6C11.

In embodiment the MAb 5C10 antibody as defined herein above binds to oneor more amino acid residues of the glycosylated MUC1 epitope having thesequence VTSAPDT(Core3)RPAPGSTAPPAHG (SEQ ID NO: 24)

In embodiment the MAb 4D9 antibody as defined herein above binds to oneor more amino acid residues of the glycosylated MUC4 epitope having thesequence PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In embodiment the MAb 3C9 antibody as defined herein above binds to oneor more amino acid residues of the glycosylated MUC4 epitope having thesequence PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In embodiment the MAb 6C11 antibody as defined herein above binds to oneor more amino acid residues of the glycosylated MUC4 epitope having thesequence PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23)wherein the asterisk (*) indicates a potential O-glycosylation site,wherein the optional glycan is independently selected from the groupconsisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn(GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

In embodiment the MAb 6E3 antibody as defined herein above binds to oneor more amino acid residues of the glycosylated MUC4 epitope having thesequencePMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQID NO: 30) wherein the asterisk (*) indicates a potentialO-glycosylation site, wherein the optional glycan is independentlyselected from the group consisting of Core-3(GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn(Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4(GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T(Neu5Acα2-3Galβ3GalNAc-Ser/Thr).

Antibodies can be used as carriers for functional groups conjugated tothe antibodies. The functional groups may be for example agents suitablefor detection by imaging methods or toxins useful in killing ordestroying cells, for example cancer cells.

In one aspect the present invention relates to a method of detecting apeptide and/or an antibody as defined herein above, said methodcomprising conjugating an imaging agent to said peptide or antibody.

In one embodiment said imaging agent is detectable by at least one ofthe technologies selected from the group consisting of: computertomography, ultrasound, magnetic resonance, nuclear imaging, opticaland/or electron optical imaging.

In a further embodiment said optical and/or electron optical imaging isselected from the group consisting of diffuse optical tomography,optical coherence tomography, confocal laser scanning, microscopy,electron microscopy, fluorescence correlation microscopy, fluorescenceresonance energy transfer, and fluorescence lifetime imaging.

In a further embodiment said nuclear imaging is selected from the groupconsisting of PET, SPECT and MRI.

In one embodiment said imaging agent is selected from the groupconsisting of antibodies, small molecules, peptides and metal ions,wherein said metal ion is selected from ions of transition metals orlanthanides and actinides.

In one embodiment the metal ion is an ion of Hf, Ho or Gd.

In one embodiment the antibody of the present invention, furthercomprises a toxin conjugated to said antibody.

The antibodies of the present invention have been raised againstdifferent parts of glycosylated mucin polypeptides, Thus in one aspect,the present invention relates to a monoclonal antibody or an antigenbinding fragment of said antibody, wherein said antibody or the antigenbinding fragment of said antibody is capable of specifically recognisinga mucin glycopeptide as defined herein above.

The method used by the present inventors for raising antibodies is alsopart of the invention. Thus, in one aspect, the present inventionrelates to a method for producing the antibody defined herein above,said method comprising the steps of:

-   -   i) providing a host organism,    -   ii) immunizing the host organism with an O-glycosylated mucin as        defined herein above, and    -   iii) obtaining said antibody.

The present invention is applicable to a subject undergoing therapy,such as surgery.

In surgery the glycopeptides epitopes identified by the presentinventors can be targeted with antibodies of the invention wherein theantibodies are conjugated to a visualization label, such as afluorescent label. The epitopes can thus be visualised during surgerywith the effect of more efficiently removing e.g. cancer tumours.

Accordingly, in one aspect, the present invention relates to a methodfor detecting a cancer tumour in a patient undergoing therapy and/orexamination, said method comprising the steps of:

(i) administering to an area of the patient, the antibody as definedherein above, or an antigen binding fragment of said antibody, suitablyconjugated to a visualisation label, and

(ii) removing unbound antibodies, and

(iii) detecting antibodies bound to glycopeptide epitopes as definedherein above, wherein labelling indicates presence of a tumour.

In one embodiment of the present invention the therapy is surgery.

In one embodiment of the present invention the examination isexamination for colorectal cancer.

In one embodiment of the present invention the examination forcolorectal cancer is by visualisation means such as endoscopy.

Another aspect of the present invention is a method for the preparationof hybridoma cells, which secrete monoclonal antibodies specific for theimmunogenic glycopeptide characterized in that a suitable mammal isimmunized with the immunogenic glycopeptide, antibody-producing cells ofsaid mammal are fused with cells of a continuous cell line, the hybridcells obtained in the fusion are cloned, and cell clones secreting thedesired antibodies are selected.

Still another aspect is a monoclonal antibody selected from the groupconsisting of a monoclonal antibody produced by the hybridoma cellsprepared by the method described above, a monoclonal antibody preparedby molecular display techniques, such as mRNA display, ribosome display,phage display and covalent display against the immunogenic glycopeptide.

Traditionally, monoclonal antibodies have been prepared using hybridomatechnology. However, alternative techniques such as mRNA display,ribosome display, phage display and covalent display are now available.These are all display techniques where a peptide library is selectedagainst the immunogenic glycopeptide. Such techniques can e.g. be usedto identify humanized or fully human antibodies.

In one embodiment, the monoclonal antibody binds the MUC4 or MUC1glycopeptides specified herein, on cancer cells but not on anon-malignant counterpart. Preferably said antibody binds glycopeptideepitopes associated with colorectal cancer.

In one aspect, the present invention relates to a monoclonal antibody oran antigen binding fragment of said antibody, wherein said antibody orthe antigen binding fragment of said antibody is capable of specificallyrecognising a mucin glycopeptide as defined herein above.

Quantitative and Qualitative Analysis of Bound Material

The step of the method of the present invention comprising qualitativelyand/or quantitatively characterising the bound peptide or antibody isbased on an ELISA-type, or ELISA-analogous method. ELISA-analogousmethods may comprise using e.g. microbeads to which peptides orantibodies can be used. One example of a microbead method is the Luminexmethod (www.luminexcorp.com).

Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassayor EIA, is a biochemical technique used mainly in immunology to detectthe presence of an antibody or an antigen in a sample. The ELISA hasbeen used as a diagnostic tool in medicine and plant pathology, as wellas a quality control check in various industries. In simple terms, inELISA an unknown amount of antigen is affixed to a surface, and then aspecific antibody is washed over the surface so that it can bind to theantigen. This antibody is linked to an enzyme, and in the final step asubstance is added that the enzyme can convert to some detectablesignal. Thus in the case of fluorescence ELISA, when light of theappropriate wavelength is shone upon the sample, any antigen/antibodycomplexes will fluoresce so that the amount of antigen in the sample canbe inferred through the magnitude of the fluorescence.

Performing an ELISA involves at least one antibody with specificity fora particular antigen. The sample with an unknown amount of antigen isimmobilized on a solid support (usually a polystyrene microtiter plate)either non-specifically (via adsorption to the surface) or specifically(via capture by another antibody specific to the same antigen, in a“sandwich” ELISA). After the antigen is immobilized the detectionantibody is added, forming a complex with the antigen. The detectionantibody can be covalently linked to an enzyme, or can itself bedetected by a secondary antibody which is linked to an enzyme throughconjugation. Between each step the plate is typically washed with a milddetergent solution to remove any proteins or antibodies that are notspecifically bound. After the final wash step the plate is developed byadding an enzymatic substrate to produce a visible signal, whichindicates the quantity of antigen in the sample.

In one embodiment the step of qualitatively and/or quantitativelycharacterising bound material in the method of the present invention isby Enzyme-linked immunosorbent assay (ELISA).

In one embodiment the step of qualitatively and/or quantitativelycharacterising bound material in the method of the present invention isby a bead assay such as a Luminex assay.

Pharmaceutical Compositions and Administration Forms

The main routes of drug delivery, in the treatment method areintravenous, oral, and topical. Other drug-administration methods, suchas intraveneous, subcutaneous and intramuscular injection or viainhalation, which are effective to deliver the drug to a target site orto introduce the drug into the bloodstream, are also contemplated.

The mucosal membrane to which the pharmaceutical preparation of theinvention may be administered can be any mucosal membrane of the mammalto which the biologically active substance is to be given, e.g. in thenose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract,or rectum, preferably the mucosa of the nose, mouth or vagina.

Compounds of the invention may be administered parenterally, that is byintravenous, intramuscular, subcutaneous intranasal, intrarectal,intravaginal or intraperitoneal administration. The subcutaneous andintramuscular forms of parenteral administration are generallypreferred. Appropriate dosage forms for such administration may beprepared by conventional techniques. The compounds may also beadministered by inhalation, which is by intranasal and oral inhalationadministration. Appropriate dosage forms for such administration, suchas an aerosol formulation or a metered dose inhaler, may be prepared byconventional techniques.

The compounds according to the invention may be administered with atleast one other compound. The compounds may be administeredsimultaneously, either as separate formulations or combined in a unitdosage form, or administered sequentially.

In one embodiment of the present invention, the dosage of the activeingredient of the pharmaceutical composition as defined herein above, isbetween 10 μg to 500 mg per kg body mass.

Formulations

Whilst it is possible for the compounds or salts of the presentinvention to be administered as the raw glycopeptide or antibodypreparation, it is preferred to present them in the form of apharmaceutical formulation. Accordingly, the present invention furtherprovides a pharmaceutical formulation, for medicinal application or foruse during in situ detection of autoantibodies or glycopeptides relatingto disease. The pharmaceutical composition comprises a compound of thepresent invention or a pharmaceutically acceptable salt thereof, asherein defined, and a pharmaceutically acceptable carrier therefore.

The compounds of the present invention may be formulated in a widevariety of oral administration dosage forms. The pharmaceuticalcompositions and dosage forms may comprise the compounds of theinvention or its pharmaceutically acceptable salt or a crystal formthereof as the active component. The pharmaceutically acceptablecarriers can be either solid or liquid. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. A solid carrier can be one or more substanceswhich may also act as diluents, flavoring agents, solubilizers,lubricants, suspending agents, binders, preservatives, wetting agents,tablet disintegrating agents, or an encapsulating material.

Preferably, the composition will be about 0.5% to 75% by weight of acompound or compounds of the invention, with the remainder consisting ofsuitable pharmaceutical excipients. For oral administration, suchexcipients include pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,gelatin, sucrose, magnesium carbonate, and the like.

In powders, the carrier is a finely divided solid which is a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired. Powders and tablets preferably contain from one to aboutseventy percent of the active compound. Suitable carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as carrier providing acapsule in which the active component, with or without carriers, issurrounded by a carrier, which is in association with it. Similarly,cachets and lozenges are included. Tablets, powders, capsules, pills,cachets, and lozenges can be as solid forms suitable for oraladministration.

Drops according to the present invention may comprise sterile ornon-sterile aqueous or oil solutions or suspensions, and may be preparedby dissolving the active ingredient in a suitable aqueous solution,optionally including a bactericidal and/or fungicidal agent and/or anyother suitable preservative, and optionally including a surface activeagent. The resulting solution may then be clarified by filtration,transferred to a suitable container which is then sealed and sterilizedby autoclaving or maintaining at 98-100° C. for half an hour.Alternatively, the solution may be sterilized by filtration andtransferred to the container aseptically. Examples of bactericidal andfungicidal agents suitable for inclusion in the drops are phenylmercuricnitrate or acetate (0.002%), benzalkonium chloride (0.01%) andchlorhexidine acetate (0.01%). Suitable solvents for the preparation ofan oily solution include glycerol, diluted alcohol and propylene glycol.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavours, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Other forms suitable for oral administration include liquid formpreparations including emulsions, syrups, elixirs, aqueous solutions,aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solidform preparations which are intended to be converted shortly before useto liquid form preparations. Emulsions may be prepared in solutions inaqueous propylene glycol solutions or may contain emulsifying agentssuch as lecithin, sorbitan monooleate, or acacia. Aqueous solutions canbe prepared by dissolving the active component in water and addingsuitable colorants, flavours, stabilizing and thickening agents. Aqueoussuspensions can be prepared by dispersing the finely divided activecomponent in water with viscous material, such as natural or syntheticgums, resins, methylcellulose, sodium carboxymethylcellulose, and otherwell known suspending agents. Solid form preparations include solutions,suspensions, and emulsions, and may contain, in addition to the activecomponent, colorants, flavours, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The compounds of the present invention may be formulated for parenteraladministration (e.g., by injection, for example bolus injection orcontinuous infusion) and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, forexample solutions in aqueous polyethylene glycol. Examples of oily ornonaqueous carriers, diluents, solvents or vehicles include propyleneglycol, polyethylene glycol, vegetable oils (e.g., olive oil), andinjectable organic esters (e.g., ethyl oleate), and may containformulatory agents such as preserving, wetting, emulsifying orsuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form, obtained by aseptic isolationof sterile solid or by lyophilisation from solution for constitutionbefore use with a suitable vehicle, e.g., sterile, pyrogen-free water.

Oils useful in parenteral formulations include petroleum, animal,vegetable, or synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides; (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example,alkyl-.beta.-aminopropionates, and 2-alkyl-imidazoline quaternaryammonium salts, and (e) mixtures thereof.

The parenteral formulations typically will contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Preservativesand buffers may be used. In order to minimize or eliminate irritation atthe site of injection, such compositions may contain one or morenonionic surfactants having a hydrophile-lipophile balance (HLB) of fromabout 12 to about 17. The quantity of surfactant in such formulationswill typically range from about 5 to about 15% by weight. Suitablesurfactants include polyethylene sorbitan fatty acid esters, such assorbitan monooleate and the high molecular weight adducts of ethyleneoxide with a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

The compounds of the invention can also be delivered topically. Regionsfor topical administration include the skin surface and also mucousmembrane tissues of the vagina, rectum, nose, mouth, and throat.Compositions for topical administration via the skin and mucousmembranes should not give rise to signs of irritation, such as swellingor redness.

The topical composition may include a pharmaceutically acceptablecarrier adapted for topical administration. Thus, the composition maytake the form of a suspension, solution, ointment, lotion, sexuallubricant, cream, foam, aerosol, spray, suppository, implant, inhalant,tablet, capsule, dry powder, syrup, balm or lozenge, for example.Methods for preparing such compositions are well known in thepharmaceutical industry.

The compounds of the present invention may be formulated for topicaladministration to the epidermis as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also containing one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or colouring agents. Formulations suitable for topicaladministration in the mouth include lozenges comprising active agents ina flavoured base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatin andglycerin or sucrose and acacia; and mouthwashes comprising the activeingredient in a suitable liquid carrier.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy base. The base may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives or a fattyacid such as steric or oleic acid together with an alcohol such aspropylene glycol or a macrogel. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurfactant such as a sorbitan ester or a polyoxyethylene derivativethereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Transdermal Delivery

The pharmaceutical agent-chemical modifier complexes described hereincan be administered transdermally. Transdermal administration typicallyinvolves the delivery of a pharmaceutical agent for percutaneous passageof the drug into the systemic circulation of the patient. The skin sitesinclude anatomic regions for transdermally administering the drug andinclude the forearm, abdomen, chest, back, buttock, mastoidal area, andthe like.

Transdermal delivery is accomplished by exposing a source of the complexto a patient's skin for an extended period of time. Transdermal patcheshave the added advantage of providing controlled delivery of apharmaceutical agent-chemical modifier complex to the body. SeeTransdermal Drug Delivery: Developmental Issues and ResearchInitiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989);Controlled Drug Delivery: Fundamentals and Applications, Robinson andLee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery ofDrugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC Press, (1987). Suchdosage forms can be made by dissolving, dispersing, or otherwiseincorporating the pharmaceutical agent-chemical modifier complex in aproper medium, such as an elastomeric matrix material. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate of such flux can be controlled by either providing arate-controlling membrane or dispersing the compound in a polymer matrixor gel.

Passive Transdermal Drug Delivery

A variety of types of transdermal patches will find use in the methodsdescribed herein. For example, a simple adhesive patch can be preparedfrom a backing material and an acrylate adhesive. The pharmaceuticalagent-chemical modifier complex and any enhancer are formulated into theadhesive casting solution and allowed to mix thoroughly. The solution iscast directly onto the backing material and the casting solvent isevaporated in an oven, leaving an adhesive film. The release liner canbe attached to complete the system.

Alternatively, a polyurethane matrix patch can be employed to deliverthe pharmaceutical agent-chemical modifier complex. The layers of thispatch comprise a backing, a polyurethane drug/enhancer matrix, amembrane, an adhesive, and a release liner. The polyurethane matrix isprepared using a room temperature curing polyurethane prepolymer.Addition of water, alcohol, and complex to the prepolymer results in theformation of a tacky firm elastomer that can be directly cast only thebacking material.

A further embodiment of this invention will utilize a hydrogel matrixpatch. Typically, the hydrogel matrix will comprise alcohol, water,drug, and several hydrophilic polymers.

This hydrogel matrix can be incorporated into a transdermal patchbetween the backing and the adhesive layer.

The liquid reservoir patch will also find use in the methods describedherein. This patch comprises an impermeable or semipermeable, heatsealable backing material, a heat sealable membrane, an acrylate basedpressure sensitive skin adhesive, and a siliconized release liner. Thebacking is heat sealed to the membrane to form a reservoir which canthen be filled with a solution of the complex, enhancers, gelling agent,and other excipients.

Foam matrix patches are similar in design and components to the liquidreservoir system, except that the gelled pharmaceutical agent-chemicalmodifier solution is constrained in a thin foam layer, typically apolyurethane. This foam layer is situated between the backing and themembrane which have been heat sealed at the periphery of the patch.

For passive delivery systems, the rate of release is typicallycontrolled by a membrane placed between the reservoir and the skin, bydiffusion from a monolithic device, or by the skin itself serving as arate-controlling barrier in the delivery system. See U.S. Pat. Nos.4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062; and the like. Therate of drug delivery will be dependent, in part, upon the nature of themembrane. For example, the rate of drug delivery across membranes withinthe body is generally higher than across dermal barriers. The rate atwhich the complex is delivered from the device to the membrane is mostadvantageously controlled by the use of rate-limiting membranes whichare placed between the reservoir and the skin. Assuming that the skin issufficiently permeable to the complex (i.e., absorption through the skinis greater than the rate of passage through the membrane), the membranewill serve to control the dosage rate experienced by the patient.

Suitable permeable membrane materials may be selected based on thedesired degree of permeability, the nature of the complex, and themechanical considerations related to constructing the device. Exemplarypermeable membrane materials include a wide variety of natural andsynthetic polymers, such as polydimethylsiloxanes (silicone rubbers),ethylenevinylacetate copolymer (EVA), polyurethanes,polyurethane-polyether copolymers, polyethylenes, polyamides,polyvinylchlorides (PVC), polypropylenes, polycarbonates,polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulosetriacetate and cellulose nitrate/acetate, and hydrogels, e.g.,2-hydroxyethylmethacrylate (HEMA).

Other items may be contained in the device, such as other conventionalcomponents of therapeutic products, depending upon the desired devicecharacteristics. For example, the compositions according to thisinvention may also include one or more preservatives or bacteriostaticagents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate,chlorocresol, benzalkonium chlorides, and the like. These pharmaceuticalcompositions also can contain other active ingredients such asantimicrobial agents, particularly antibiotics, anesthetics, analgesics,and antipruritic agents.

The compounds of the present invention may be formulated foradministration as suppositories. A low melting wax, such as a mixture offatty acid glycerides or cocoa butter is first melted and the activecomponent is dispersed homogeneously, for example, by stirring. Themolten homogeneous mixture is then poured into convenient sized molds,allowed to cool, and to solidify.

The active compound may be formulated into a suppository comprising, forexample, about 0.5% to about 50% of a compound of the invention,disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%]and PEG 4000 [4%].

The compounds of the present invention may be formulated for vaginaladministration. Pessaries, tampons, creams, gels, pastes, foams orsprays containing in addition to the active ingredient such carriers asare known in the art to be appropriate.

The compounds of the present invention may be formulated for nasaladministration. The solutions or suspensions are applied directly to thenasal cavity by conventional means, for example with a dropper, pipetteor spray. The formulations may be provided in a single or multidoseform. In the latter case of a dropper or pipette this may be achieved bythe patient administering an appropriate, predetermined volume of thesolution or suspension. In the case of a spray this may be achieved forexample by means of a metering atomizing spray pump.

The compounds of the present invention may be formulated for aerosoladministration, particularly to the respiratory tract and includingintranasal administration. The compound will generally have a smallparticle size for example of the order of 5 microns or less. Such aparticle size may be obtained by means known in the art, for example bymicronization. The active ingredient is provided in a pressurized packwith a suitable propellant such as a chlorofluorocarbon (CFC) forexample dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetrafluoroethane, carbon dioxide or other suitable gas. Theaerosol may conveniently also contain a surfactant such as lecithin. Thedose of drug may be controlled by a metered valve. Alternatively theactive ingredients may be provided in a form of a dry powder, forexample a powder mix of the compound in a suitable powder base such aslactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). The powder carrier will form agel in the nasal cavity. The powder composition may be presented in unitdose form for example in capsules or cartridges of e.g., gelatin orblister packs from which the powder may be administered by means of aninhaler.

When desired, formulations can be prepared with enteric coatings adaptedfor sustained or controlled release administration of the activeingredient.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

Pharmaceutically Acceptable Salts

Pharmaceutically acceptable salts of the instant compounds, where theycan be prepared, are also intended to be covered by this invention.These salts will be ones which are acceptable in their application to apharmaceutical use. By that it is meant that the salt will retain thebiological activity of the parent compound and the salt will not haveuntoward or deleterious effects in its application and use in treatingdiseases.

Pharmaceutically acceptable salts are prepared in a standard manner. Ifthe parent compound is a base it is treated with an excess of an organicor inorganic acid in a suitable solvent. If the parent compound is anacid, it is treated with an inorganic or organic base in a suitablesolvent.

The compounds of the invention may be administered in the form of analkali metal or earth alkali metal salt thereof, concurrently,simultaneously, or together with a pharmaceutically acceptable carrieror diluent, especially and preferably in the form of a pharmaceuticalcomposition thereof, whether by oral, rectal, or parenteral (includingsubcutaneous) route, in an effective amount.

Examples of pharmaceutically acceptable acid addition salts for use inthe present inventive pharmaceutical composition include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric and sulfuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, forexample.

Therapeutically useful agents can be formulated into a composition withthe antibodies of the invention and need not be directly attached to theantibodies of the invention. However, in some embodiments,therapeutically useful agents are attached to the antibodies of theinvention using methods available to one of skill in the art, forexample, standard coupling procedures.

Compositions may contain antibodies, antigenic epitopes or trypsin-likeprotease inhibitors. Such compositions are useful for detecting theantigenic peptide epitopes (glycopeptides) and for therapeutic methodsinvolving prevention and treatment of cancers associated with thepresence of said antigenic epitopes.

The antibodies, (and for example antigenic epitopes and proteaseinhibitors) can be formulated as pharmaceutical compositions andadministered to a mammalian host, such as a human patient in a varietyof forms adapted to the chosen route of administration. Routes foradministration include, for example, intravenous, intra-arterial,subcutaneous, intramuscular, intraperitoneal and other routes selectedby one of skill in the art.

Solutions of the antibodies, (and for example antigenic epitopes andprotease inhibitors) can be prepared in water or saline, and optionallymixed with a nontoxic surfactant. Formulations for intravenous orintra-arterial administration may include sterile aqueous solutions thatmay also contain buffers, liposomes, diluents and other suitableadditives.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions comprising the activeingredient that are adapted for administration by encapsulation inliposomes. In all cases, the ultimate dosage form must be sterile, fluidand stable under the conditions of manufacture and storage.

Sterile injectable solutions are prepared by incorporating theantibodies, antigenic epitopes and protease inhibitors in the requiredamount in the appropriate solvent with various of the other ingredientsenumerated above, as required, followed by filter sterilization.

In one aspect the present invention relates to a pharmaceuticalcomposition comprising an active ingredient, wherein said activeingredient is selected from (i) one or more of the O-glycosylatedpeptides defined herein above, or (ii) the antibody capable ofrecognising the peptide of (i), or any other antibody defined hereinabove, and wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier or diluent.

In one embodiment the pharmaceutical composition is formulated foradministration by injection, suppository, oral administration,sublingual tablet or spray, cutaneous administration or inhalation.

In one embodiment said injection is intramuscular, intravenous,intranasal, intraperitoneal, subcutaneous, a bolus or a continuousadministration.

In one embodiment the pH of the pharmaceutical composition is between pH4 and pH 10.

In one embodiment administration of the pharmaceutical compositionoccurs at intervals of 30 minutes to 24 hours.

In one embodiment administration of the pharmaceutical compositionoccurs at intervals of 1 to 6 hours.

In one embodiment the duration of the treatment with the pharmaceuticalcomposition is from 6 to 72 hours.

In one embodiment the duration of the treatment with the pharmaceuticalcomposition is life long.

In one embodiment the dosage of the active ingredient is between 10 μgto 500 mg per kg body mass.

Diagnostic Kits

Kits for detection of the antigenic peptide epitopes of the presentinvention can be provided.

A kit for detection of the antigenic epitope of the invention maycontain a container containing an antibody capable of binding to anantigenic epitope of the invention. Such an antibody may be labeled foreasy detection. Individual kits may be adapted for performing one ormore of the methods of the invention. Optionally, the subject kit mayfurther comprise at least one other reagent required for performing themethod that the kit is adapted to perform. Examples of such additionalreagents include: a label, a standard, a control, a buffer, a solutionfor diluting the test sample, or a reagent that facilitates detection ofthe label. The reagents included in the kits of the invention may besupplied in premeasured units so as to provide for greater precision andaccuracy. Typically, kits reagents and other components are placed andcontained in separate vessels. A reaction vessel, test tube, microwelltray, microtiter dish or other container can also be included in thekit. Different labels can be used on different reagents so that eachreagent can be distinguished from another.

The kit can also be for the treatment of cancer comprising apharmaceutical composition and an instructional material. Such a kit maycontain a container having an antigenic epitope, an antibody or aninhibitor of the invention. The antigenic epitope may act as a vaccinefor preventing formation of metastatic adenocarcinoma. The antibody isdirected against an antigenic epitope of the invention and can beadministered to treat or prevent the spread of adenocarcinomas. Any oneof these antigenic epitopes, antibodies or inhibitors may be containedwithin an appropriate container in the kit. Alternatively, a combinationof antigenic epitopes, antibodies or inhibitors may be contained withinan appropriate container in the kit.

Further, a kit comprising a pharmaceutical composition and a deliverydevice for delivering the composition to a mammal, for example, a humanpatient who may have an adenocarcinoma can also be provided. By way ofexample, the delivery device may be a squeezable spray bottle, ametered-dose spray bottle, an aerosol spray device, an atomizer, a drypowder delivery device, a self-propelling solvent/powder-dispensingdevice, a syringe, a needle, a tampon, or a dosage measuring container.

In one aspect, the present invention relates to a method for identifyinga disease (e.g. cancer such as colorectal cancer) associated withshedding of O-glycosylated peptides or peptide fragments, said methodcomprising the steps of:

-   -   (i) selecting potential target polypeptides containing potential        O-glycosylation sites, and    -   (ii) producing recombinant fragments covering specific areas of        interest from each potential target, and/or    -   (iii) producing synthetic peptides covering specific areas of        interest from each potential target, and    -   (iv) in vitro glycosylate the fragments of (ii) and/or (iii)        using recombinant glycosyltransferases    -   (v) purifying the fragments of (iv), and    -   (vi) characterizing the purified products of (v), and    -   (vii) printing of non-glycosylated and glycosylated targets,    -   (viii) screening the printed targets of (vii) with sera from a        potentially diseased sample host and    -   (ix) screening the printed targets of (vii) with sera from a        healthy sample host as control,    -   wherein the presence of auto-antibodies bound to the printed        targets of (viii) indicates disease in the potentially diseased        sample host.

In one embodiment of the method for identifying a disease defined hereinabove, the recombinant fragments covering specific areas of interestfrom each potential target are between 10 to 30 kDa.

In one embodiment of the method for identifying a disease defined hereinabove, the synthetic fragments covering specific areas of interest fromeach potential target are between 10 and 30 amino acid residues.

In one embodiment of the method for identifying a disease defined hereinabove, the purification is by HPLC.

In one embodiment of the method for identifying a disease defined hereinabove, the characterization of the glycosylation products is byMALDI-TOF.

In one embodiment the kit of the invention comprises items useful in themethod defined herein above.

Device

The glycopeptides of the invention have been used to construct a devicefor detecting disease, in particular cancer and especially colorectalcancer. One way of constructing the device is the Mucin O-glycopeptidearray print method demonstrated in example 2.

Thus, in one aspect the method defined herein, wherein the at least two,or the one or more O-glycosylated peptides are conjugated to a surfaceform a glycopeptide array device discussed above.

In one aspect the invention is directed to a device comprising at leasttwo different O-glycosylated peptides conjugated to a surface, whereinthe at least two different peptides are selected from the peptidesdefined herein above.

In an analogous embodiment the method is reversed such that theantibodies defined herein above are conjugated to a surface thus formingan antibody array device of the present invention.

In one such embodiment the invention relates to a device comprising aplurality of different antibodies conjugated to a surface, wherein oneor more of the antibodies is/are selected from the antibodies definedherein above

In a further embodiment the invention relates to the use of the devicedefined herein above.

In another aspect, the present invention relates to a device comprisinga plurality of glycosylated peptides attached to a surface, wherein atleast a part of the peptides are selected from the peptides definedherein above.

As discussed above, the glycopeptides and the antibodies of theinvention act in a lock-and-key manner. Thus, in one aspect, the presentinvention relates to a device comprising a plurality of antibodiescovalently attached to a surface, wherein at least a part of theantibodies are selected from the antibodies as defined herein, i.e.including but not limited to the antibodies selected from the groupconsisting of MAb 5C10, MAb 3C9, MAb 4D9, MAb 6C11 and MAb 6E3.

In one aspect, the present invention relates to a mucin peptide asdefined herein above for use as a medicament, in particular for use in amethod of treatment of cancer. Said mucin peptide used in said method oftreatment of cancer can e.g. include immunisation of an individual byadministering to said individual the glycosylated mucin peptide. Themedical use also applies to the other part of the lock-and-keyinvention.

Thus, in this aspect the present invention relates to a method ofimmunising an individual, by administering to said individual anantibody of the invention

In one aspect, the present invention relates to a device comprising aplurality of glycosylated peptides attached to a surface, wherein atleast a part of the peptides are selected from the peptides definedherein above.

In a further aspect, the present invention relates to a devicecomprising a plurality of antibodies covalently attached to a surface,wherein at least a part of the antibodies are selected from theantibodies defined herein above.

In a further embodiment, the device comprises a mixture of glycosylatedpeptides and antibodies according to the present invention, and may thusbe used as a multi-detection tool.

In one aspect the invention relates to a device comprising a pluralityof glycosylated peptides conjugated to a surface, wherein at least apart of the peptides are selected from the peptides defined hereinabove.

In one aspect the invention relates to a device comprising a pluralityof antibodies conjugated to a surface, wherein at least a part of theantibodies are selected from the antibodies defined herein above.

In one aspect the present invention relates to the use of the devicedefined herein above, in a method of identifying auto-antibodiesassociated with disease, said method comprising contacting said devicewith a sample from a host organism.

In one aspect the present invention relates to the use of the devicedefined herein above in a method of identifying O-glycosylated peptidesassociated with disease, said method comprising contacting said devicewith a sample from a host organism.

In one embodiment the disease identifiable using said device is cancer,wherein the cancer is selected from the group consisting of colorectalcancer, breast cancer, oral cancer, gastric cancer, esophageal cancer,pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer,renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer,basal cell cancer, squamous cell cancer, malignant melanoma, bladdercancer, endometrial cancer and cervix cancer.

In one embodiment the disease identifiable using said device iscolorectal cancer.

Methods of Treating Cancer

Passive Immunisation

Passive immunity is the transfer of active humoral immunity in the formof readymade antibodies, such as the antibodies of the presentinvention. Passive immunity can occur naturally, when maternalantibodies are transferred to the fetus through the placenta, and canalso be induced artificially, when high levels of human antibodiesspecific for e.g. an O-glycosylated mucin peptide of the invention istransferred to non-immune individuals.

One method of treating cancer, such as colorectal cancer is passiveimmunisation.

In one aspect the antibody defined herein can be used in a method oftreatment of cancer comprising passive immunisation. The cancer isselected from the group consisting of colorectal cancer, breast cancer,oral cancer, gastric cancer, esophageal cancer, pancreatic cancer,cholangiocarcinoma, ovarian cancer, lung cancer, renal cancer, prostatecancer, hepatocellular carcinoma, testis cancer, basal cell cancer,squamous cell cancer, malignant melanoma, bladder cancer, endometrialcancer and cervix cancer.

In one embodiment, one or more antibodies of the invention have beenconjugated to one or more toxins capable of destroying the cells such ascancer cells.

A toxin is understood as an agent having cytotoxic properties. A toxinmay be e.g. a biotoxin such as a toxin produced by microorganisms or aprotein isolated from the venom of the cone snail, spider, snake,scorpion, jellyfish, wasp, bee, ant, termite, honeybee, wasp, poisondart frog.

In one embodiment the toxin is selected from the group consisting ofcyanotoxins, hemotoxins, necrotoxins, cytotoxins such as but not limitedto ricin and apitoxin.

In one embodiment the toxin conjugated to the antibody of the inventionis selected from the group consisting of small molecules, metals, metalions, small inorganic molecules, proteins, peptides, glycopeptides, RNA,DNA and siRNA.

In one embodiment the toxin conjugated to the antibody of the inventionis a venom from spider, snake, scorpion, jellyfish, wasp, bee, ant,termite, honeybee, wasp or poison dart frog.

In one embodiment the toxin conjugated to the antibody of the inventionis selected from the group consisting of cyanotoxin, hemotoxin,necrotoxin and cytotoxin.

In one embodiment a toxin has been conjugated to the antibody of theinvention as defined herein above.

In one embodiment the toxin-conjugated antibodies of the invention areadministered to a patient afflicted with disease, such as cancer.

In one embodiment, the combination of toxins is such that individuallythey are not potent cell killers, but when presented simultaneously toan individual cell their combined effects are lethal to that cell.

In one embodiment the toxin conjugated antibody of the invention targetsa cancer cell that presents a matching O-glycosylated mucin peptideepitope on the cell surface, resulting in that the conjugated toxin canact to destroy the cancer cell presenting the O-glycosylated mucinpeptide epitope on the cell surface.

In a further embodiment of the invention, one or more antibodies capableof recognising different O-glycosylated mucin targets presented by cellsof a particular cancer are administered to the patient; these antibodiescan similarly be conjugated to one or more toxins capably of destroyingthe cancer cells. In a particular embodiment, the combination of toxinsis such that individually they are not potent cell killers, but whenpresented simultaneously to an individual cell their combined effectsare lethal to that cell.

In a further aspect, the present invention relates to a method ofpassively immunising an individual, said method comprising administeringto said individual an antibody as defined herein above.

Active Immunisation

In one aspect, also the glycosylated mucin peptides as defined hereinabove may be used as a medicament e.g. in a method of treatment ofcancer, wherein the cancer is selected from the group consisting ofcolorectal cancer, breast cancer, oral cancer, gastric cancer,esophageal cancer, pancreatic cancer, cholangiocarcinoma, ovariancancer, lung cancer, renal cancer, prostate cancer, hepatocellularcarcinoma, testis cancer, basal cell cancer, squamous cell cancer,malignant melanoma, bladder cancer, endometrial cancer and cervixcancer.

In one aspect, the present invention relates to a method of activelyimmunizing an individual, said method comprising administering to saidindividual a glycosylated mucin peptide as defined herein above.

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EXAMPLES Example 1 Sequences of the Invention

Overview of Sequences

SEQ ID NO: 1 MUC4

SEQ ID NO: 2 MUC1

SEQ ID NO: 3 MUC4 peptide

SEQ ID NO: 4 MUC4 peptide

SEQ ID NO: 5 MUC4 peptide

SEQ ID NO: 6 MUC4 peptide

SEQ ID NO: 7 MUC4 peptide

SEQ ID NO: 8 MUC4 peptide

SEQ ID NO: 9 MUC4 peptide

SEQ ID NO: 10 MUC4 peptide

SEQ ID NO: 11 MUC4 peptide

SEQ ID NO: 12 MUC4 peptide

SEQ ID NO: 13 MUC4 peptide

SEQ ID NO: 14 MUC4 peptide

SEQ ID NO: 15 MUC4 peptide

SEQ ID NO: 16 MUC4 peptide

SEQ ID NO: 17 MUC4 peptide

SEQ ID NO: 18 MUC4 peptide

SEQ ID NO: 19 MUC4 peptide

SEQ ID NO: 20 MUC4 peptide

SEQ ID NO: 21 MUC4 peptide

SEQ ID NO: 22 MUC4 peptide

SEQ ID NO: 23 MUC4 peptide

SEQ ID NO: 24 MUC1 peptide

SEQ ID NO: 25 MUC2 peptide

SEQ ID NO: 26 MUC2

SEQ ID NO: 27 MUC5AC

SEQ ID NO: 28 MUC6

SEQ ID NO: 29 MUC7

SEQ ID NO: 30 recMUC4 peptide

SEQ ID NO: 31 recMUC1 peptide

SEQ ID NO: 32 recMUC2 peptide

SEQ ID NO: 33 recMUC5AC peptide

SEQ ID NO: 34 recMUC6 peptide

SEQ ID NO: 35 recMUC7 peptide

SEQ ID NO: 36 MUC3

SEQ ID NO: 37 MUC3B

SEQ ID NO: 38 MUC5B

SEQ ID NO: 39 MUC8

SEQ ID NO: 40 MUC12

SEQ ID NO: 41 MUC13

SEQ ID NO: 42 MUC14

SEQ ID NO: 43 MUC15

SEQ ID NO: 44 MUC16

SEQ ID NO: 45 MUC17

SEQ ID NO: 46 MUC19

SEQ ID NO: 47 MUC20

SEQ ID NO: 48 MUC 21

SEQ ID NO: 49 MUC HEG

SEQ ID NO: 50 MUC9

SEQ ID NO: 51 MUC18

SEQ ID NO: 52 p53 peptide

SEQ ID NO: 53 p53 peptide

SEQ ID NO: 54 p53 peptide

SEQ ID NO: 55 p53 peptide

SEQ ID NO: 56 p53 peptide

SEQ ID NO: 57 p53 peptide

SEQ ID NO: 58 p53 peptide

SEQ ID NO: 59 p53 peptide

SEQ ID NO: 60 p53 peptide

SEQ ID NO: 61 p53 peptide

SEQ ID NO: 62 p53 peptide

SEQ ID NO: 63 p53 peptide

SEQ ID NO: 64 p53 peptide

SEQ ID NO: 65 p53 peptide

SEQ ID NO: 66 p53 peptide

SEQ ID NO: 67 p53 peptide

SEQ ID NO: 68 p53 peptide

SEQ ID NO: 69 p53 peptide

Sequence list SEQ ID NO: 1 (MUC4; human)  >sp|Q99102|MUC4_HUMAN Mucin-4 OS =Homo Sapiens GN = MUC4 PE = 1 SV = 2MKGARWRRVPWVSLSCLCLCLLPHVVPGTTEDTLITGSKTPAPVTSTGSTTATLEGQSTAASSRTSNQDISASSQNHQTKSTETTSKAQTDILTQMMTSTLFSSPSVHNVMETVTQETAPPDEMTTSFPSSVTNTLMMTSKTITMTTSTDSTLGNTEETSTAGTESSTPVTSAVSITAGQEGQSRTTSWRTSIQDTSASSQNHWTRSTQTTRESQTSTLTHRTTSTPSFSPSVHNVTGTVSQKTSPSGETATSSLCSVTNTSMMTSEKITVTTSTGSTLGNPGETSSVPVTGSLMPVTSAALVTVDPEGQSPATFSRTSTQDTTAFSKNHQTQSVETTRVSQINTLNTLTPVTTSTVLSSPSGFNPSGTVSQETFPSGETTISSPSSVSNTFLVTSKVFRMPISRDSTLGNTEETSLSVSGTISAITSKVSTIWWSDTLSTALSPSSLPPKISTAFHTQQSEGAETTGRPHERSSFSPGVSQEIFTLHETTTVVPSSFSSKGHTTWSQTELPSTSTGAATRLVTGNPSTRAAGTIPRVPSKVSAIGEPGEPTTYSSHSTTLPKTTGAGAQTQWTQETGTTGEALLSSPSYSVIQMIKTATSPSSSPMLDRHTSQQITTAPSTNHSTIHSTSTSPQESPAVSQRGHTRAPQTTQESQTTRSVSPMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATTFAPAPTGNGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFSSNPSRDSHTTQSTTELLSASASHGAIPVSTGMASSIVPGTFHPTLSEASTAGRPTGQSSPTSPSASPQETAAISRMAQTQRTGTSRGSDTISLASQATDTFSTVPPTPPSITSSGLTSPQTQTHTLSPSGSGKTFTTALISNATPLPVTSTSSASTGHATPLAVSSATSASTVSSDSPLKMETSGMTTPSLKTDGGRRTATSPPPTTSQTIISTIPSTAMHTRSTAAPIPILPERGVSLFPYGADAGDLEFVRRTVDFTSPLFKPATGFPLGSSLRDSLYFTDNGQIIFPESDYQIFSYPNPLPTGFTGRDPVALVAPFWDDADFSTGRGTTFYQEYETFYGEHSLLVQQAESWIRKITNNGGYKARWALKVTWVNAHAYPAQWTLGSNTYQAILSTDGSRSYALFLYQSGGMQWDVAQRSGNPVLMGFSSGDGYFENSPLMSQPVWERYRPDRFLNSNSGLQGLQFYRLHREERPNYRLECLQWLKSQPRWPSWGWNQVSCPCSWQQGRRDLRFQPVSIGRWGLGSRQLCSFTSWRGGVCCSYGPWGEFREGWHVQRPWQLAQELEPQSWCCRWNDKPYLCALYQQRRPHVGCATYRPPQPAWMFGDPHITTLDGVSYTFNGLGDFLLVGAQDGNSSFLLQGRTAQTGSAQATNFIAFAAQYRSSSLGPVTVQWLLEPHDAIRVLLDNQTVTFQPDHEDGGGQETFNATGVLLSRNGSEVSASFDGWATVSVIALSNILHASASLPPEYQNRTEGLLGVWNNNPEDDFRMPNGSTIPPGSPEEMLFHFGMTWQINGTGLLGKRNDQLPSNFTPVFYSQLQKNSSWAEHLISNCDGDSSCIYDTLALRNASIGLHTREVSKNYEQANATLNQYPPSINGGRVIEAYKGQTTLIQYTSNAEDANFTLRDSCTDLELFENGTLLWTPKSLEPFTLEILARSAKIGLASALQPRTVVCHCNAESQCLYNQTSRVGNSSLEVAGCKCDGGTFGRYCEGSEDACEEPCFPSVHCVPGKGCEACPPNLTGDGRHCAALGSSFLCQNQSCPVNYCYNQGHCYISQTLGCQPMCTCPPAFTDSRCFLAGNNFSPTVNLELPLRVIQLLLSEEENASMAEVNASVAYRLGTLDMRAFLRNSQVERIDSAAPASGSPIQHWMVISEFQYRPRGPVIDFLNNQLLAAVVEAFLYHVPRRSEEPRNDVVFQPISGEDVRDVTALNVSTLKAYFRCDGYKGYDLVYSPQSGFTCVSPCSRGYCDHGGQCQHLPSGPRCSCVSFSIYTAWGEHCEHLSMKLDAFFGIFFGALGGLLLLGVGTFVVLRFWGCSGARFSYFLNSAEALPSEQ ID NO: 2 (MUC1; human)  >sp|P15941|MUC1_HUMAN Mucin-1 OS =Homo Sapiens GN = MUC1 PE = 1 SV = 2MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVISAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL SEQ ID NO: 3 (human MUC4 peptide)  PMTDTKTVTTPGSSFTA SEQ ID NO: 4 (human MUC4 peptide)  PGSSFTASGHSPSEIVPQD SEQ ID NO: 5 (human MUC4 peptide)  SEIVPQDAPTISAATTFAPA SEQ ID NO: 6 (human MUC4 peptide)  TTFAPAPTGNGHTTQAPTTA SEQ ID NO: 7 (human MUC4 peptide)  TTQAPTTALQAAPSSHD SEQ ID NO: 8 (human MUC4 peptide)  APSSHDATLGPSGGTSLSKT SEQ ID NO: 9 (human MUC4 peptide)  SLSKTGALTLANSVVSTP SEQ ID NO: 10 (human MUC4 peptide)  NSVVSTPGGPEGQWTSASAS SEQ ID NO: 11 (human MUC4 peptide)  TSASASTSPRTAAAMTHT SEQ ID NO: 12 (human MUC4 peptide)  AAAMTHTHQAESTEASGQT SEQ ID NO: 13 (human MUC4 peptide)  EASGQTQTSEPASSGSRTT SEQ ID NO: 14 (human MUC4 peptide)  PASSGSRTTSAGTATPSSS SEQ ID NO: 15 (human MUC4 peptide)  TATPSSSGASGTTPSGSEGI SEQ ID NO: 16 (human MUC4 peptide)  SGSEGISTSGETTRFSSN SEQ ID NO: 17 (human MUC4 peptide)  GETTRFSSNPSRDSHTT SEQ ID NO: 18 (human MUC4 peptide)  PVTSPSSASTGHTTPLPVTDTSSASTGDTTP SEQ ID NO: 19 (human MUC4 peptide)  LPVTSLSSVSTGDTTPLPVTS PSSASTGH SEQ ID NO: 20 (human MUC4 peptide)  LPVTSPSSASTGHASPLLVTDASSASTGQ SEQ ID NO: 21 (human MUC4 peptide)  PLPVTSPSSASTGHASPLLVTDASSASTGQ SEQ ID NO: 22 (human MUC4 peptide)  STGDTLPLPVTDTSSV SEQ ID NO: 23 (human MUC4 peptide)  PVTYASSASTGDTTPLPVTDTSSVSTGHAT SEQ ID NO: 24 (human MUC1 peptide)  VTSAPDTRPAPGSTAPPAHG SEQ ID NO: 25 (human MUC2 peptide)  PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC SEQ ID NO: 26 (MUC2 human)  >sp|Q02817|MUC2_HUMAN Mucin-2 OS =Homo Sapiens GN = MUC2 PE = 1 SV = 2MGLPLARLAAVCLALSLAGGSELQTEGRTRYHGRNVCSTWGNFHYKTFDGDVFRFPGLCDYNFASDCRGSYKEFAVHLKRGPGQAEAPAGVESILLTIKDDTIYLTRHLAVLNGAVVSTPHYSPGLLIEKSDAYTKVYSRAGLTLMWNREDALMLELDTKFRNHTCGLCGDYNGLQSYSEFLSDGVLFSPLEFGNMQKINQPDVVCEDPEEEVAPASCSEHRAECERLLTAEAFADCQDLVPLEPYLRACQQDRCRCPGGDTCVCSTVAEFSRQCSHAGGRPGNWRTATLCPKTCPGNLVYLESGSPCMDTCSHLEVSSLCEEHRMDGCFCPEGTVYDDIGDSGCVPVSQCHCRLHGHLYTPGQEITNDCEQCVCNAGRWVCKDLPCPGICALEGGSHITTFDGKTYTFHGDCYYVLAKGDHNDSYALLGELAPCGSTDKQTCLKTVVLLADKKKNAVVFKSDGSVLLNQLQVNLPHVTASFSVFRPSSYHIMVSMAIGVRLQVQLAPVMQLFVTLDQASQGQVQGLCGNFNGLEGDDFKTASGLVEATGAGFANTWKAQSTCHDKLDWLDDPCSLNIESANYAEHWCSLLKKTETPFGRCHSAVDPAEYYKRCKYDTCNCQNNEDCLCAALSSYARACTAKGVMLWGWREHVCNKDVGSCPNSQVFLYNLTTCQQTCRSLSEADSHCLEGFAPVDGCGCPDHTFLDEKGRCVPLAKCSCYHRGLYLEAGDVVVRQEERCVCRDGRLHCRQIRLIGQSCTAPKIHMDCSNLTALATSKPRALSCQTLAAGYYHTECVSGCVCPDGLMDDGRGGCVVEKECPCVHNNDLYSSGAKIKVDCNTCTCKRGRWVCTQAVCHGTCSIYGSGHYITFDGKYYDFDGHCSYVAVQDYCGQNSSLGSFSIITENVPCGTTGVTCSKAIKIFMGRTELKLEDKHRVVIQRDEGHHVAYTTREVGQYLVVESSTGIIVIWDKRTTVFIKLAPSYKGTVCGLCGNFDHRSNNDFTTRDHMVVSSELDFGNSWKEAPTCPDVSTNPEPCSLNPHRRSWAEKQCSILKSSVFSICHSKVDPKPFYEACVHDSCSCDTGGDCECFCSAVASYAQECTKEGACVFWRTPDLCPIFCDYYNPPHECEWHYEPCGNRSFETCRTINGIHSNISVSYLEGCYPRCPKDRPIYEEDLKKCVTADKCGCYVEDTHYPPGASVPTEETCKSCVCTNSSQVVCRPEEGKILNQTQDGAFCYWEICGPNGTVEKHFNICSITTRPSTLTTFTTITLPTTPTSFTTTTTTTTPTSSTVLSTTPKLCCLWSDWINEDHPSSGSDDGDREPFDGVCGAPEDIECRSVKDPHLSLEQHGQKVQCDVSVGFICKNEDQFGNGPFGLCYDYKIRVNCCWPMDKCITTPSPPTTTPSPPPTTTTTLPPTTTPSPPTTTTTTPPPTTTPSPPITTTTTPLPTTTPSPPISTTTTPPPTTTPSPPTTTPSPPTTTPSPPTTTTTTPPPTTTPSPPMTTPITPPASTTTLPPTTTPSPPTTTTTTPPPTTTPSPPTTTPITPPTSTTTLPPTTTPSPPPTTTTTPPPTTTPSPPTTTTPSPPTITTTTPPPTTTPSPPTTTTTTPPPTTTPSPPTTTPITPPTSTTTLPPTTTPSPPPTTTTTPPPTTTPSPPTTTTPSPPITTTTTPPPTTTPSSPITTTPSPPTTTMTTPSPTTTPSSPITTTTTPSSTTTPSPPPTTMTTPSPTTTPSPPTTTMTTLPPTTTSSPLTTTPLPPSITPPTFSPFSTTTPTTPCVPLCNWTGWLDSGKPNFHKPGGDTELIGDVCGPGWAANISCRATMYPDVPIGQLGQTVVCDVSVGLICKNEDQKPGGVIPMAFCLNYEINVQCCECVTQPTIMITTTTENPTPPITTPITTTITVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITITTIVIPTPTPTGTQTPTTTPITTTTIVTPTPTPTGTQTPTTTPITTTTIVIPTPTPTGTQTPTTTPITTTTIVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITITTIVIPTPTPTGTQTPTTTPITTTTIVTPTPTPTGTQTPTTTPITTTTIVIPTPTPTGTQTPTTTPITTTTIVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTITTVTPTPTPTGTQTGPPTHTSTAPIAELTTSNPPPESSTPQTSRSTSSPLTESTTLLSTLPPAIEMTSTAPPSTPTAPTTTSGGHTLSPPPSTTTSPPGTPTRGTTTGSSSAPTPSTVQTTTTSAWTPTPTPLSTPSIIRTTGLRPYPSSVLICCVLNDTYYAPGEEVYNGTYGDTCYFVNCSLSCTLEFYNWSCPSTPSPTPTPSKSTPTPSKPSSTPSKPTPGTKPPECPDFDPPRQENETWWLCDCFMATCKYNNTVEIVKVECEPPPMPTCSNGLQPVRVEDPDGCCWHWECDCYCTGWGDPHYVTFDGLYYSYQGNCTYVLVEEISPSVDNFGVYIDNYHCDPNDKVSCPRTLIVRHETQEVLIKTVHMMPMQVQVQVNRQAVALPYKKYGLEVYQSGINYVVDIPELGVLVSYNGLSFSVRLPYHRFGNNTKGQCGTCTNTTSDDCILPSGEIVSNCEAAADQWLVNDPSKPHCPHSSSTTKRPAVTVPGGGKTTPHKDCTPSPLCQLIKDSLFAQCHALVPPQHYYDACVFDSCFMPGSSLECASLQAYAALCAQQNICLDWRNHTHGACLVECPSHREYQACGPAEEPTCKSSSSQQNNTVLVEGCFCPEGTMNYAPGFDVCVKTCGCVGPDNVPREFGEHFEFDCKNCVCLEGGSGIICQPKRCSQKPVTHCVEDGTYLATEVNPADTCCNITVCKCNTSLCKEKPSVCPLGFEVKSKMVPGRCCPFYWCESKGVCVHGNAEYQPGSPVYSSKCQDCVCTDKVDNNTLLNVIACTHVPCNTSCSPGFELMEAPGECCKKCEQTHCIIKRPDNQHVILKPGDFKSDPKNNCTFFSCVKIHNQLISSVSNITCPNFDASICIPGSITFMPNGCCKTCTPRNETRVPCSTVPVTTEVSYAGCTKTVLMNHCSGSCGTFVMYSAKAQALDHSCSCCKEEKTSQREVVLSCPNGGSLTHTYTHIESCQCQDTVCGLPTGTSRRARRSPRHLGSGSEQ ID NO: 27 (MUC5AC, human)  >tr|O75372|O75372_HUMAN Gastric mucin (Fragment) OS =Homo Sapiens GN = MUC5AC PE = 1SV = 1MSVGRRKLALLWALALALACTRHTGHAQDGSSESSYKHHPALSPIARGPIGVPLRGATVFPSLRTIPVVRASNPAHNGRVCSTWGSFHYKTFDGDVFRFPGLCNYVFSEHCGAAYEDFNIPATPQPGVSGPHAEQGPHEGGWRGHPADQGLRPGQRPPGPAALQPVWGPHSARAAATPRWKPGWALSSCGTTMTACCWKLDTKYANKNLWALWGLQRDARGQRAPLPQHQADTHGIREPAERWTNPRSSVRTLSLNPRRTAPLALASCEELLHGQLFSGCVALVDVGSYLEACRQDLCFCEDTDLLSCVCHTLAEYSRQCTHAGGLPQDWRGPDFCPQKCPNNMQYHECRSPCADTCSNQEHSRACEDHCVAGCFCPEGTVLDDIGQTGCVPVSKCACVYNGAAYAPGATYSTDCTNCTCSGGRWSCQEVPCPGTCSVLGGAHFSTFDGKQYTVHGDCSYVLTKPCDSSAFTVLAELRRCGLTDSETCLKSVTLSLDGAQTVVVIKASGEVFLNQIYTQLPISAANVTIFRPSTFFIIAQTSLGLQLNLQLVPTMQLFMQLAPKLRGQTCGLCGNFNSIQADDFRTLSGVVEATAAAFFNTFKTQAACPNIRNSFEDPCSLSVENEKYAQHWCSQLTDADGPFGRCHAAVKPGTYYSNCMFDTCNCERSEDCLCAALSSYVHACAAKGVQLGGWRDGVCTKPMTTCPKSMTYHYHVSTCQPTCRSLSEGDITCSVGFIPVDGCICPKGTFLDDTGKCVQASNCPCYHRGSMIPNGESVHDSGAICTCTHGKLSCIGGQAPAPVCAAPMVFFDCRNATPGDTGAGCQKSCHTLDMTCYSPQCVPGCVCPDGLVADGEGGCITAEDCPCVHNEASYRAGQTIRVGCNTCTCDSRMWRCTDDPCLATCAVYGDGHYLTFDGQSYSFNEETASTRWCRTAVAGKTAPRTPFVLSPRTSPAAPQGPPAPRPSRFSWGNFELKLSHGKVEVIGTDESQEVPYTIRQMGIYLVVDTDIGLVLLWDKKTSIFINLSPEFKGRVCGLCGNFDDIAVNDFATRSRSVVGDVLEFGNSWKLSPSCPDALAPKDPCTANPFRKSWAQKQCSILHGPTFAACHAHVEPARYYEACVNDACACDSGGDCECFCTAVARYAQACHEVGTCVCLRTPSICPLFCDYYNPEGQCEWHYQPCGVPCLRTCRNPRGDCLRDVRGLEGCYPKCPPEAPIFDEDKMQCVATCPTPPLPPRCHVHGKSYRPGAVVPSDKNCQSCLCTERGVECTYKAEACVCTYNGQRFHPGDVIYHTTDGTGGCISARCGANGTIERRVYPCSPTTPVPPTTFSFSTPPLVVSSTHTPSNGPSSAHTGPPSSAWPTTAGTSPRTSEQ ID NO: 28 (MUC6, human)  >sp|Q6W4X9|MUC6_HUMAN Mucin-6 OS =Homo Sapiens GN = MUC6 PE = 1 SV = 2MVQRWLLLSCCGALLSAGLANTSYTSPGLQRLKDSPQTAPDKGQCSTWGAGHFSTFDHHV YDFSGTCNYIFAATCKDAFPSFSVQLRRGPDGSISRIIVELGASVVIVSEAIISVKDIGV ISLPYTSNGLQITPFGQSVRLVAKQLELELEVVWGPDSHLMVLVERKYMGQMCGLCGNFD GKVTNEFVSEEGKFLEPHKFAALQKLDDPGEICTFQDIPSTHVRQAQHARGCTQLLTLVA PECSVSKEPFVLSCQADVAAAPQPGPQNSSYATLSEYSRQCSMVGQPVALRSPGLCSVGQ CPANQVYQECGSACVKTCSNSEHSCSSSCTFGCFCPEGTDLNDLSNNHTCVPVTQCPCVL HGAMYAPGEVTIAACQTCRCTLGRWVCTERPCPGHCSLEGGSFVTTFDARPYRFHGTCTY ILLQSPQLPEDGALMAVYDKSGVSHSETSLVAVVYLSRQDKIVISQDEVVTNNGEAKWLP YKTRNITVFRQTSTHLQMATSFGLELVVQLRPIFQAYVTVGPQFRGQTRGLCGNFNGDTT DDFTTSMGIAEGTASLFVDSWRAGNCPDALERETDPCSMSQLNKVCAETHCSMLLRTGTV FERCHATVNPAPIYKRCMYQACNYEETFPHICAALGDYVHACSLRGVLLWGWRSSVDNCT IPCTGNTTFSYNSQACERTCLSLSDRATECHHSAVPVDGCNCPDGTYLNQKGECVRKAQC PCILEGYKFILAEQSTVINGITCHCINGRLSCPQRLQMPLASCQAPKTFKSCSQSSENKF GAACAPTCQMLATGVACVPTKCEPGCVCAEGLYENAYGQCVPPEECPCEFSGVSYPGGAE LHTDCRTCSCSRGRWACQQGTHCPSTCTLYGEGHVITFDGQRFVFDGNCEYILATDVCGV NYSQPTFKILTENVICGNSGVICSRAIKIFLGGLSVVLADRNYTVTGEEPHVQLGVTPGA LSLVVDISIPGRYNLTLIWNRHMTILIRIARASQDPLCGLCGNFNGNMKDDFETRSRYVA SSELELVNSWKESPLCGDVSFVTDPCSLNAFRRSWAERKCSVINSQTFATCHSKVYHLPY YEACVRDACGCDSGGDCECLCDAVAAYAQACLDKGVCVDWRTPAFCPIYCGFYNTHTQDG HGEYQYTQEANCTWHYQPCLCPSQPQSVPGSNIEGCYNCSQDEYFDHEEGVCVPCMPPTT PQPPTTPQLPTTGSRPTQVWPMTGTSTTIGLLSSTGPSPSSNHTPASPTQTPLLPATLTS SKPTASSGEPPRPTTAVTPQATSGLPPTATLRSTATKPTVTQATTRATASTASPATTSTA QSTTRTTMTLPTPATSGTSPTLPKSTNQELPGITATQTTGPRPTPASTTGPTTPQPGQPT RPTATETTQTRTTTEYTTPQTPHTTHSPPTAGSPVPSTGPVTATSFHATTTYPTPSHPET TLPTHVPPFSTSLVTPSTHTVITPTHAQMASSASNHSAPTGTIPPPTTLKATGSTHTAPP ITPTTSGTSQAHSSFSTNKTPTSLHSHTSSTHHPEVTPTSTTSITPNPTSTRTRTPMAHT NSATSSRPPPPFTTHSPPTGSSPFSSTGPMTATSFKTTTTYPTPSHPQTTLPTHVPPFST SLVTPSTHTVITPTHAQMATSASIHSMPTGTIPPPTTLKATGSTHTAPTMTLTTSGTSQA LSSLNTAKTSTSLHSHTSSTHHAEATSTSTTNITPNPTSTGTPPMTVTTSTRTPVAHTTS ATSSRLPTPFTTHSPPTGTTPISSTGPVTATSFQTTTTYPTPSHPHTTLPTHVPSFSTSL VTPSTHTVIIPTHTQMATSASIHSMPTGTIPPPTTIKATGSTHTAPPMTPTTSGTSQSPS SFSTAKTSTSLPYHTSSTHHPEVTPTSTTNITPKHTSTGTRTPVAHTTSASSSRLPTPFT THSPPTGSSPFSSTGPMTATSFQTTTTYPTPSHPQTTLPTHVPPFSTSLVTPSTHTVIIT THTQMATSASIHSTPTGTVPPPTTLKATGSTHTAPPMTVTTSGTSQTHSSFSTATASSSF ISSSSWLPQNSSSRPPSSPITTQLPHLSSATTPVSTTNQLSSSFSPSPSAPSTVSSYVPS SHSSPQTSSPSVGTSSSFVSAPVHSTTLSSGSHSSLSTHPTTASVSASPLFPSSPAASTT IRATLPHTISSPFTLSALLPISTVTVSPTPSSHLASSTIAFPSTPRTTASTHTAPAFSSQ STTSRSTSLTTRVPTSGFVSLTSGVTGIPTSPVTNLTTRHPGPTLSPTTRFLTSSLTAHG STPASAPVSSLGTPTPTSPGVCSVREQQEEITFKGCMANVTVTRCEGACISAASFNIITQ QVDARCSCCRPLHSYEQQLELPCPDPSTPGRRLVLTLQVFSHCVCSSVACGD SEQ ID NO: 29 (MUC7, human)  >sp|Q8TAX7|MUC7_HUMAN Mucin-7 OS =Homo Sapiens GN = MUC7 PE = 1 SV = 1MKTLPLFVCICALSACFSFSEGRERDHELRHRRHHHQSPKSHFELPHYPGLLAHQKPFIRKSYKCLHKRCRPKLPPSPNNPPKFPNPHQPPKHPDKNSSVVNPTLVATTQIPSVTFPSASTKITTLPNVTFLPQNATTISSRENVNTSSSVATLAPVNSPAPQDTTAAPPTPSATTPAPPSSSAPPETTAAPPTPSATTQAPPSSSAPPETTAAPPTPPATTPAPPSSSAPPETTAAPPTPSATTPAPLSSSAPPETTAVPPTPSATTLDPSSASAPPETTAAPPTPSATTPAPPSSPAPQETTAAPITTPNSSPTTLAPDTSETSAAPTHQTITSVTTQTTTTKQPTSAPGQNKISRFLLYMKNLLNRIIDDMVEQ SEQ ID NO: 30 (recMUC4) PMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGNGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFSSNPSRDSHTT SEQ ID NO: 31 (recMUC1) MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRDPNSVTSAPDTRPAPGSTAPQAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDT SEQ ID NO: 32 (recMUC2) MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRDPNSSSVDKLDIEFLQPGGSVQCCECVTQPTTMTTTTTENPTPTPITTTTTVTPTPTPTSTQSTTPTPITTTNTVTPTPTPTGTQTSEQ ID NO: 33 (recMUC5AC) MGSSHHHHHHSSGLVPRGSHMASMIGGQQMGRIPTSSITSTPQTSTTSASTTSITSGPGTTPSPVPITSTTSAPTTSTTSAATTSTISAPTTSTTSAPTTSTTSASTASKTSGLGTTPSPIPTTSTTSPPTTSTTSASTASKTSGPGTTPSPVPTTSTIFAPRTSTTSASTTSTTPGPGTTPSPVPTTSTASVSKTSTSHVSISKTTHSQAAALEHHHHHH SEQ ID NO: 34 (recMUC6) MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIRSTSLVTPSTHTVITPTHAQMATSASNHSAPTGTIPPPTTLKATGSTHTAPPITPTTSGTSQAHSSFSTNKAAALEHHHHHH SEQ ID NO: 35 (recMUC7) MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIPAPQDTTAAPPTPSATTPAPPSSSAPPETTAAPPTPSATTQAPPSSSAPPETTAAPPTPPATTPAPPSSSAPPETTAAPPTPSATTPAPLSSSAPPETTAVPPTPSATTLDPSSASAPPETTAAPPTPSATTPAPPSSPAPQETTAAPITTPNSSPTTLAPDTSETSAAPTHQTTTSVTTQTTTTKQPTSAPAAALEHHHHHH SEQ ID NO: 36 (hMUC3) MQLLGLLGLLWMLKASPWATGTLSTATSISQVPFPRAEAASAVLSNSPHSRDLAGWPLGVPQLASPAPGHRENAPMTLTTSPHDTLISETLLNSPVSSNTSTTPTSKFAFKVETTPPTVLVYSATTECVYPTSFIITISHPTSICVTTTQVAFTSSYTSTPVTQKPVTTVTSTYSMTTTEKGTSAMTSSPSTTTARETPIVTVTPSSVSATDTTFHTTISSTTRTTERTPLPTGSIHTTTSPTPVFTTLKTAVTSTSSITSSITSTNTVTSMTTTASQPTATNTLSSPTRTILSSTPVLSTETITSGITNTTPLSTLVTTLPTTISRSTPTSETTYPTSPXXXXXXXXXXAMTSPPPVSSSITPTNTMTSMRTTTYWPTATNTLSPLTSSILSSTPVPSTEMITSHTTNTTPLSTLVTTLLTTITRSTPTSETTYPTSPTSIVSDSTTEITYSTSITGTLSTATTLPPTSSSLPTTETATMTPTTTLITTTPNTTSHSTPSFTSSTIYSTVSTSTTAISSASPTSGTMVTSTTMTPSSLSTDTPSTTPTTITYPSVGSTGFLTTATDLTSTFTVSSSSAMSKSVIPSSPSIQNTETSSLVSMTSATTPSLRPTITSTDSTLTSSLLTTFPSTYSFSSSMSASSAGTTHTETISSLPASTNTIHTTAESALAPTTTTSFTTSPTMEPPSTIVATTGTGQTTFPSSTATFLETTILTPTTDFSTESLTTAMTSTPPITSSITPTDTMTSMRTTTSWPTATNTLSPLTSSILSSTPVPSTEVTTSHTTNTNPVSTLVTTLPITITRSTLTSETAYPSSPTSTVTESTTEITYPTTMTETSSTATSLPPTSSLVSTAETAKTPTTNLXXXXXXXXXXFTSSTSLLHSQHTTXLPSPSVPTTLGTMVTSTSXIPSSLSTDIPTSQPTTITPSSVGITGSLPMMTDLTSVYTVSSMSARPTSVIPSSPTVQNTETSIFVSMMSATTPSGGSTFTSTENTPTRSLLTSFPVTHSFSSSMSASSVGTTHTQSISSPPAITSTLHTTAESTPSPTTTMSFTTFTKMETPSSTVATTGTGQTTFTSSTATSPKTTTLIPTSDISTGSFKTAVSSTPPITSSITSTYTVTSMTTTTPLGPTATNTLPSFTSSVSSSTPVPSTEAITSGTTNTTPLSTLVTTFSNSDTSSTPTSETTYPTSLTSALTDSTTRTTYSTNMTGTLSTVTSLRPTSSSLLTTVTATVPTTNLVTTTTKITSHSTPSFTSSIATTETPSHSTPRFTSSITTTETPSHSTPRFTSSITNTKTTSHSSPSFTSSITTTDSIVXXXXXXXXXXITITETTSHSTPSYTTSITTTETPSHSTPSYTTSITTTETPSHSTPSFTSSITTTETTSHSTPSFTSSIRTTETTSYSTPSFTSSNTITETTSHSTPSYITSITTTETPSSSTPSFSSSITTTETTSHSTPGFTSSITTTETTSHSTPSFTSSITTTETTSHDTPSFTSSITTSETPSHSTPSSTSLITTTKTTSHSTPSFTSSITTTETTSHSARSFTSSITTTETTSHNTRSFTSSITTTETNSHSTTSFTSSITTTETTSHSTPSFSSSITTTETPLHSTPGLPSWVTTTKTTSHITPGLTSSITTTETTSHSTPGFTSSITTTETTSESTPSLSSSTIYSTVSTSTTAITSHFTTSETAVTPTPVTPSSLSTDIPTTSLRTLTPSSVGTSTSLTTTTDFPSIPTDISTLPTRTHIISSSPSIQSTETSSLVGTTSPTMSTVRMTLRITENTPISSFSTSIVVIPETPTQTPPVLTSATGTQTSPAPTTVTFGSTDSSTSTLHTLTPSTALSTIVSTSQVPIPSTHSSTLQTTPSTPSLQTSLTSTSEFTTESFTRGSTSTNAILTSFSTIIWSSTPTIIMSSSPSSASITPVFSTTIHSVPSSPYIFSTENVGSASITGFPSLSSSATTSTSSTSSSLTTALTEITPFSYISLPSTTPCPGTITITIVPASPTDPCVEMDPSTEATSPPTTPLTVFPFTTEMVTCPTSISIQTTLTTYMDTSSMMPESESSISPNASSSTGTGTVPTNTVFTSTRLPTSETWLSNSSVIPLPLPGVSTIPLTMKPSSSLPTILRTSSKSTHPSPPTTRTSETPVATTQTPTTLTSRRTTRITSQMTTQSTLTTTAGTCDNGGTWEQGQCACLPGFSGDRCQLQTRCQNGGQWDGLKCQCPSTFYGSSCEFAVEQVDLDVVETEVGMEVSVDQQFSPDLNDNTSQAYRDFNKTFWNQMQKIFADMQGFTFKGVEILSLRNGSIVVDYLVLLEMPFSPQLESEYEQVKTTLKEGLQNASQDANSCQDSQTLCFKPDSIKVNNNSKTELTPEAICRRAAPTGYEEFYFPLVEATRLRCVTKCTSGVDNAIDCHQGQCVLETSGPACRCYSTDTHWFSGPRCEVAVHWRALVGGLTAGAALLVLLLLALGVRAVRSGWWGGQRRGRSWDQDRKWFETWDEEVVGIFSNWGFEDDGTDKDINFHVALENVDTTMKVHIKRPEMTSSSV SEQ ID NO: 37 (hMUC3B) MQLLGLLSILWMLKSSPGATGTLSTATSTSHVTFPRAEATRTALSNSPHSRYLAEWPQGVPQLASPAPGHRENAPMTLTTSPHDTLISETLLSSLVSSNTSTTPTSKFAFKVETTPPTVLVYSATTECVYPTSFIITISHSTSICVTTTQVTFTSSYTPTPVTQKPVTTVTRTYPMTTTEKGTSAMISSPSTTTARETPIVTVTPSSSVSATDTTFHTTISSTTRTTERTPLPTGSIHTTMSPTPVFTTLKTAVTSTSPITSTITSTNTVTSMTTTTSRPTATNTLSSLTSSILSSTPAPNTEVITSHTTTTTPPSTLVTTLPTAIARSTPTSETTXXXXXXXXXXTIYSTVSSSTTAITSPFTTAETGVTSTPSSPSSLSTDIPTTSLRTLTPLSLSTSTSLTTTTDLPSIPTDISSLPTPIHIISSSPSIQSTETSSLVGTTSPTMSTVRATLRSTENTPISSFSTSIVVTPETPTTQAPPVLMSATGTQTSPVPTTVTFGSMDSSTSTLHTLTPSTALSKIMSTSQFPIPSTHSSTLQTTPSIPSLQTSLTSTSEFTTESFTRGSTSTNAILTSFSTIIWSSTPTIIMSSSPSSASITPVFATTIHSVPSSPYIFSTENVGSASITAFPSLSSSSTTSTSPTSSSLTTALTEITPFSYISLPSTTPCPGTITITIVPASPTDPCVEMDPSTEATSPPTTPLTVFPFTTEMVTCPSSISMQTTLATHMDTSSMTPESESSIIPNASSSTGIGTVPINTVFTSTRLPTSETWLSNNSVIPTPLPGVSTIPLTMKPSSSLPTILRTSSKSTHPSPPTARTSETSVATTQTPTTLTTRRTTPITSWMTTQSTLTTTAGTCDNGGTWEQGQCACLPGFSGDRCQLQTRCQNGGQWDGLKCQCPSTFYGSSCEFAVEQVDLDVVETEVGMEVSVDQQFSPDLNDNTSQAYRDFNKTFWNQMQKIFADMQGFTFKGVEILSLRNGSIVVDYLVLLEMPFSPQLESEYEQVKTTLKEGLQNASQDANSCQDSQTLCFKPDSIKVNNNSKTELTPEAICRRAAPTGYEEFYFPLVEATRLRCVTKCTSGVDNAIDCHQGQCVLETSGPACRCYSTDTHWFSGPRCEVAVHWRALVGGLTAGAALLVLLLLALGVRAVRSGWWGGQRRGRSWDQDRKWFETWDEEVVGTFSNWGFEDDGTDKDTNFHVALENVDTTMKVHIKRPEMTSSSV SEQ ID NO: 38 (hMUC5B) MGAPSACRTLVLALAAMLVVPQAETQGPVEPSWGNAGHTMDGGAPTSSPTRRVSFVPPVTVFPSLSPLNPAHNGRVCSTVVGDPHYKTFDGDVFRFPGLCNYVFSEHCRAAYEDFNVQLRRGLVGSRPVVIRVVIKAQGLVLEASNGSVLINGQREELPYSRTGLLVEQSGDYIKVSIRLVLTFLWNGEDSALLELDPKYANQTCGLCGDFNGLPAFNEFYAHNARLTPLQFGNLQKLDGPTEQCPDPLPLPAGNCTDEEGICHRTLLGPAFAECHALVDSTAYLAACAQDLCRCPTCPCATFVEYSRQCAHAGGQPRNWRCPELCPRTCPLNMQHQECGSPCTDTCSNPQRAQLCEDHCVDGCFCPPGSTVLDDITHSGCLPLGQCPCTHGGRTYSPGTSFNTTCSSCTCSGGLWQCQDLPCPGTCSVQGGAHISTYDEKLYDLHGDCSYVLSKKCADSSFTVLAELRKCGLTDNENCLKAVTLSLDGGDTAIRVQADGGVFLNSIYTQLPLSAANITLFTPSSFFIVVQTGLGLQLLVQLVPLMQVFVRLDPAHQGQMCGLCGNFNQNQADDFTALSGVVEATGAAFANTVVKAQAACANARNSFEDPCSLSVENENYARHWCSRLTDPNSAFSRCHSIINPKPFHSNCMFDTCNCERSEDCLCAALSSYVHACAAKGVQLSDWRDGVCTKYMQNCPKSQRYAYVVDACQPTCRGLSEADVTCSVSFVPVDGCTCPAGTFLNDAGACVPAQECPCYAHGTVLAPGEVVHDEGAVCSCTGGKLSCLGASLQKSTGCAAPMVYLDCSNSSAGTPGAECLRSCHTLDVGCFSTHCVSGCVCPPGLVSDGSGGCIAEEDCPCVHNEATYKPGETIRVDCNTCTCRNRRWECSHRLCLGTCVAYGDGHFITFDGDRYSFEGSCEYILAQDYCGDNTTHGTFRIVTENIPCGTTGTTCSKAIKLFVESYELILQEGTFKAVARGPGGDPPYKIRYMGIFLVIETHGMAVSWDRKTSVFIRLHQDYKGRVCGLCGNFDDNAINDFATRSRSVVGDALEFGNSWKLSPSCPDALAPKDPCTANPFRKSWAQKQCSILHGPTFAACRSQVDSTKYYEACVNDACACDSGGDCECFCTAVAAYAQACHDAGLCVSWRTPDTCPLFCDFYNPHGGCEWHYQPCGAPCLKTCRNPSGHCLVDLPGLEGCYPKCPPSQPFFNEDQMKCVAQCGCYDKDGNYYDVGARVPTAENCQSCNCTPSGIQCAHSLEACTCTYEDRTYSYQDVIYNTTDGLGACLIAICGSNGTIIRKAVACPGTPATTPFTFTTAWVPHSTTSPALPVSTVCVREVCRWSSWYNGHRPEPGLGGGDFETFENLRQRGYQVCPVLADIECRAAQLPDMPLEELGQQVDCDRMRGLMCANSQQSPPLCHDYELRVLCCEYVPCGPSPAPGTSPQPSLSASTEPAVPTPTQTTATEKTTLWVTPSIRSTAALTSQTGSSSGPVTVTPSAPGTTTCQPRCQWTEWFDEDYPKSEQLGGDVESYDKIRAAGGHLCQQPKDIECQAESFPNWTLAQVGQKVHCDVHFGLVCRNWEQEGVFKMCYNYRIRVLCCSDDHCRGRATTPPPTTELETATTTTTQALFSTPQPTSSPGLTRAPPASTTAVPTLSEGLTSPRYTSTLGTATTGGPRQSAGSTEPTVPGVATSTLPTRSALPGTTGSLGTWRPSQPPTLAPTTMATSRARPTGTASTASKEPLITSLAPTLTSELSTSQAETSTPRTETTMSPLTNTITSQGTTRCQPKCEWTEWFDVDFPTSGVASGDMETFENIRAAGGKMCWAPKSIECRAENYPEVSIDQVGQVLTCSLETGLTCKNEDQTGRFNMCFNYNVRVLCCDDYSHCPSTLATSSTATPSSTPGTTWILTKPTTTATTTASTGSTATASSTQATAGTPHVSTTATTPTVISSKATPFSSPGTATALPALRSTATTPTATSFTAIPSSSLGTIVVTRLSQTTIPMATMSTATPSSTPETVHTSTVLTTTATTTGATGSVATPSSTPGTAHTTKVLTTTTTGFTATPSSSPGRARTLPVWISTTTTPTTRGSTVTPSSIPGTTHIPTVLITTITTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTTPIPPVLTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLSPSSPHTVCTAWTSATSGILGTTHITEPSTGTSHTPAATTGTTQHSTPALSSPHPSSRTTESPPSPGTTTPGHTTATSRTTATATPSKTRTSTLLPSQPTSAPITTVVTMGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSSTPGTTWILTELTTTATTTESTGSTATPTSTLRTAPPPKVLTTTATTPTVTSSKATPSSSPGTATALPALRSTATTPTATSVTPIPSSSLGTTWTRLSQTTTPTATMSTATPSSTPETAHTSTVLTATATTTGATGSVATPSSTPGTAHTTKVPTTTTTGFTATPSSSPGTALTPPVWISTTTTPTTRGSTVTPSSIPGTTHTATVLTTTTTTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTRPIPPVLTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLSPSSPHTVRTAWTSATSGTLGTTHITEPSTGTSHTPAATTGTTQHSTPALSSPHPSSRTTESPPSPGTTTPGHTTATSRTTATATPSKTRTSTLLPSSPTSAPITTVVTMGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSSTPGTTWILTEQTTAATTTATTGSTAIPSSTPGTAPPPKVLTSQATTPTATSSKATSSSSPRTATTLPVLTSTATKSTATSFTPIPSSTLGTTGTSQNRPPHPMATMSTIHPSSTPETTHTSTVLTTKATTTRATSSMSTPSSTPGTTWILTELTTAATTTAALPHGTPSSTPGTTWILTEPSTTATVTVPTGSTATASSTRATAGTLKVLTSTATTPTVISSRATPSSSPGTATALPALRSTATTPTATSVTAIPSSSLGTAWTRLSQTTTPTATMSTATPSSTPETVHTSTVLTTTATTTRTGSVATPSSTPGTAHTTKVPTTTTTGFTATPSSSPGTALTPPVWISTTTTPTTRGSTVTPSSIPGTTHTATVLTTTTTTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTTPIFPVLTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLPPSSPHTVPTAWTSATSGILGTTHITEPSTGTSHTPAATTGTTQPSTPALSSPHPSSRTTESPPSPGTTTPGHTRGTSRTTATATPSKTRTSTLLPSSPTSAPITTVVTTGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSSTPGTTWILTKLTTTATTTESTGSTATPSSTQGPPAGTPHVSTTATTPTVTSSKATPFSSPGTATALPALRSTATTPTATSFTAIPSSSLGTTVVTRLSQTTTPMATMSTATPSSTPETVHTSTVLTTTATTTGATGSVATPSSTPGTAHTTKVPTTTTTGFTVTPSSSPGTARTPPVWISTTTTPTTSGSTVTPSSIPGTTHTPTVLTTTTQPVATGSMATPSSSTQTSGTPPSLITTATTITATGSTTNPSSTPGTTPIPPELTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLSPSSPHTVRTAWTSATSGTLGTTHITEPSTGTSHTPAATTGTTTTSTPALSSPHPSSRTTESPPSPGTTTPGHTTATSRTTATATPSKTRTSTLLPSQPTSAPITTVVTTGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLGELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTAMPSSTPGTTWILTELTTTATTTASTGSTATPSSTPGTAPPPKVLTSPATTPTATSSKATSSSSPRTATTLPVLTSTATKSTATSVTPIPSSTLGTTGTLPEQTTTPVATMSTIHPSSTPETTHTSTVLTTKATTRATSSTSTPSSTPGTTWILTELTTAATTTAGTGPTATPSSTPGTTWILTELTTTATTTASTGSTATLSSTPGTTWILTEPSTTATVTVPTGSTATASSTQATAGTPHVSTTATTPTVTSSKATPSSSPGTATALPALRSTATTPTATSFTAIPSSSLGTTWTRLSQTTTPTATMSTATPSSTPETVHTSTVLTTTATTTGATGSVATPSSTPGTAHTTKVPTTTTTGFTATPSSSPGTALTPPVWISTTTTPTTTTPTTSGSTVTPSSIPGTTHTARVLTTTTTTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTTPIPPVLTSMATTPAATSSKATSSSSPRTATTLPVLTSTATKSTATSFTPIPSSTLWTTWTVPAQTTTPMSTMSTIHTSSTPETTHTSTVLTTTATMTRATNSTATPSSTLGTTRILTELTTTATTTAATGSTATLSSTPGTTWILTEPSTIATVMVPTGSTATTSSTLGTAHTPKVVTAMATMPTATASTVPSSSTVGTTRTPAVLPSSLPTFSVSTVSSSVLTTLRPTGFPSSHFSTPCFCRAFGQFFSPGEVIYNKTDRAGCHFYAVCNQHCDIDRFQGACPTSPPPVSSAPLSSPSPAPGCDNAIPLRQVNETWTLENCTVARCVGDNRVVLLDPKPVANVTCVNKHLPIKVSDPSQPCDFHYECECICSMWGGSHYSTFDGTSYTFRGNCTYVLMREINARFGNLSLYLDNHYCTASATAAAARCPRALSIHYKSMDIVLTVTMVHGKEEGLILFDQIPVSSGFSKNGVLVSVLGTTTMRVDIPALGVTVTFNGQVFQARLPYSLFHNNTEGQCGTCTNNQRDDCLQRDGTTAASCKDMAKTWLVPDSRKDGCWAPTGTPPTASPAAPVSSTPTPTPCPPQPLCDLMLSQVFAECHNLVPPGPFFNACISDHCRGRLEVPCQSLEAYAELCRARGVCSDWRGATGGLCDLTCPPTKVYKPCGPIQPATCNSRNQSPQLEGMAEGCFCPEDQILFNAHMGICVQACPCVGPDGFPKFPGERWVSNCQSCVCDEGSVSVQCKPLPCDAQGQPPPCNRPGFVTVTRPRAENPCCPETVCVCNTTTCPQSLPVCPPGQESICTQEEGDCCPTFRCRPQLCSYNGTFYGVGATFPGALPCHMCTCLSGDTQDPTVQCQEDACNNTTCPQGFEYKRVAGQCCGECVQTACLTPDGQPVQLNETWVNSHVDNCTVYLCEAEGGVHLLTPQPASCPDVSSCRGSLRKTGCCYSCEEDSCQVRINTTILWHQGCETEVNITFCEGSCPGASKYSAEAQAMQHQCTCCQERRVHEETVPLHCPNGSAILHTYTHVDECGCTPFCVPAPMAPPHTRGFPAQEATAVSEQ ID NO: 39 (MUC8 [Homo sapiens]) MLEFWAQVLPRDQPPSLEFWAQVLPREGLLWEPSLTEESSWATCWMGSPWEGWWFWGWFWGWFLTDAYLPACGPGCVSPGARITVTGHPTQDSEPPWGLPGPWAELAARTPPPTLPLTSWCGAGWTSCACLVFQFVCSHICPLLPRAGWGALEGRDCSGKETSRPVACRARKWREWLGCPDGGRQPRVAQASAVWQVQLKNWIIHFTYRLQSKIQKPVGLVATMLDREVLDRLAWAPASAVPPTQNILGKGFNVLPSSRLQSGGGVAMRRPAVGGVKAPGCVRIGVRVNGGLRPTIGGRCEHTQLSPPQGSPPLTSEVLWGWSGTTLREPWVGMHHSQGPGTIRAVGGCSSSPPAPPHPLPQLLPAKTPFLSTLLEAHSSCPQLLPSPEGSALWPHHQPRSLSCSRHTPQLSSFQPARVPFLLTSHPRALILPTRPGQSPEWCYEHRWAPGWHISSSIVATGLCFRESFPAICKALCKVNGCEIVFKIQFGATDPNEALEISQAICPLGFKLACGVTAKKEKVCSISCSPQPHLRAGCQLQPSACTPLPSRNGTSVYFGCIVCSCGCRLVTQQVLRPHASLEKEQRNVSQLLLGERCVTNSPWALSDNALLTGLQGDKRGFASSFGLWLGPAIIHWPLSVGGFIRSFSLSGSGGRAEPYKPRRSPCLKPHVVSVKEVSRLGPASTGQRSRAFCSGGVRWLSLSCRRPRRHQSGGLFLALQLECALSHMSRQLSVWSPQRAPFQSSCSRGCAVAWFRACCSSAGRPLPVHPGEQACSLRRGLRPAVLSPGPSLSPKPQPNATPSRGRWWLCVRWALLSSREFLRIFIARWLWVRADHRKPAGTLESRSGMQKAEGATRSSQVTLLPSCDCCRPPVPAPQRAWAVPGLQHRGLQPPPAIAPPSLCQEHWSLPVSSSGRYVTVMMSVSSHAPGSPRYHTYLSLLAATPWPSLESVSAGHAPYAVRECTSLHRCSHQKGTLQVGRRVLVPGPGPRVCPLQWGAQPGVGSGCGKEERPGTSSRDELGLFPLKVAEMQTQSSPYLWGSRQAPASGRAGFRQQPTPRGVPDRLRLQAGLDSGSTPYPVGFLTGPGFRQGWIQAAPRTRWGSRHALASGRAGFRQHPVPAGVPDMPRLQAGLDSGSTPYPVGFLTGPGFRQGWIQAAPRTRWGSRHALASAGLDSGTGVPAASALSGELRGSMSTGKIEFYEALSKVPSPLRMGSSGCQVPAARGCEAVTDRPRSTETTDRGGRGGPQNQMGTDTVGGQVPTRGPPASLDQHRGSSLHPTYPLPRSVPATPRSSLDIVSRLPHTSLRGPGSSLDTFLLLVPHCWFLGPSCSHLPCPAGPGYVPVMSGLRPGYVQVMSRLCSGYVLVRSRLCPGQVRVRFRLFLGYVWLGPGYVRVMSQLGPGYVQVVSGLGPGYFQVMSRLGPGYVRVTSRLRPGYVQVRSRLGPGYVALGPGQVRVMSGLCPGQVPVISGLCPGQVLLCSGYFQVRSQLGLGASVSHCCVPAMANLSVCGRERSHSSQLCRSAAGLPSTPGRSHARLEPGRLCFPMRDQGTGEPGLGGIRQCLQGRLGKMPLCDEERRTHKDVIRFSSCPLAPLLALNAALGAYAPESCGSLFALMRQRAPGGRANVLTQWGRKTETTEWGAGGLDQLPPDISSGKELSVQFELLFVGCSVTCILWIWPLAATGQHQFGETKTCLYSGREAFNVGTGTQMSEGLCGHRGHEGAGELGTLSVPTGLDTRGLGCSRANGTGHEGAGTLPGPMGLALVTCRAGFLAASVQVFPEETAVCVRVAEVKMTCPQCGWHLPAGVWREQKQKRGMCLSPRQLGCTLPLLPSDRTAGAPAFGLKDSHWQPRVSRLWPRAESRHQLPGFGCLLTWTEPHYRHPVLSSLQTACCGTSQPPSSGSPNRPCSLLSLCHVGSVSLETPDKHSCAGGEGSEEAVLWVTVCPQYHSLESQPPEPQNVTLLEIGSLQMKLQMLKGSSQIWLVSTSKHWPPCEKTHRPRGDGVWGREQRLQGRNHTGGKANTTRNRKRHGSSPGASGGSVAPITLVLDIRSLNLKGRHFCASSCTFFFFLTASYSVAQAGVQCRDLSSPPSLPPGFKRFSCLSLPSSWVYRCSPLYLVNFCIFSKDRVSPCWSGWSGTPDVKGSTRLGLSNCEDYRREPLHPTFTALCYGAWETTTGCSKKGSFYPTQMRAQQDSLREAHSAQGPGLLMGGGRWVPGGAASLVWRPNSFPSSWQGHLEAQLQGSLGHTGCGRPARSCTVPQGGGAARRKCQGPGASWTVWKFLFPECIISPSKIPQTQTKPTEADQRVGCSSSGGNGPGSQCLSGGHRGHKGCQPGRNQCTSTTSCPRPLQEGTRVHELPTSSPGRDPGPRAAHVLSRKGPGSTSCPRPLQEGTPGSRAAHALSRRGHRVHELPTSSPGGDTGFMSCPRPFQEGTPGSRAAHVLSRKGPRVHELPTSSPGRDPGFTSCPRPLQEGTRVTNCPRPLQEGTPGSRAAHVLSRRGHRVHELPTPSPGRDPGFMSCPRPLQEGTRVTNCPRPLQEGTRVTSCPRRLQEGTRVTSCPRPLQEGTRVTNCPRALQEGTPGSRAAHALSRKGPRVHELPTSSPGGDTGFTSCPRPLQEGTPGSRAAHALSRRGHRVHELPTSSPGRDPGHELPTSSPGGDTGFTSCPRTFQEGTPGSGLLPAHIVPLCKSEE SEQ ID NO: 40 (hMUC12) MLVIWILTLALRLCASVTIVTPEGSAVHKAISQQGTLWTGEVLEKQTVEQGKSTLRRQKNHFHRSAGELRCRNALKDEGASAGWSVMFAGESVVVLVHLWMTGARVKNLGLVEFASPGDDGDGRAEGFSLGLPLSEQARAAGAREKERQETVINHSTFSGFSQITGSTVNTSIGGNTTSASTPSSSDPFTTFSDYGVSVTFITGSTATKHFLDSSTNSGHSEESTVSHSGPGATGTTLFPSHSATSVFVGEPKTSPITSASMETTALPGSTTTAGLSEKSTTFYSSPRSPDRTLSPARTTSSGVSEKSTTSHSRPGPTHTIAFPDSTTMPGVSQESTASHSIPGSTDTTLSPGTTTPSSLGPESTTFHSSPGYTKTTRLPDNTTTSGLLEASTPVHSSTGSPHTTLSPSSSTTHEGEPTTFQSWPSSKDTSPAPSGTTSAFVKLSTTYHSSPSSTPTTHFSASSTTLGHSEESTPVHSSPVATATTPPPARSATSGHVEESTAYHRSPGSTQTMHFPESSTTSGHSEESATFHGSTTHTKSSTPSTTAALAHTSYHSSLGSTETTHFRDSSTISGRSEESKASHSSPDAMATTVLPAGSTPSVLVGDSTPSPISSGSMETTALPGSTTKPGLSEKSTTFYSSPRSPDTTHLPASMTSSGVSEESTTSHSRPGSTHTTAFPGSTTMPGLSQESTASHSSPGPTDTTLSPGSTTASSLGPEYTTFHSRPGSTETTLLPDNTTASGLLEASMPVHSSTRSPHTTLSPAGSTTRQGESTTFHSWPSSKDTRPAPPTTTSAFVEPSTTSHGSPSSIPTTHISARSTTSGLVEESTTYHSSPGSTQTMHFPESDTTSGRGEESTTSHSSTTHTISSAPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSERSTTFHSSPRSPATTLSPASTTSSGVSEESTTSRSRPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGPSQESTTSHSSSGSTDTALSPGSTTALSFGQESTTFHSNPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLQGESTAFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTTFYSSPRSPTTTLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSRGSTETTVFPHSTTTSVHGEEPTTFHSRPASTHTTLFTEDSTTSGLTEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTKLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTSDLSQEPTTSHSSQGSTEATLSPGSTTASSLGQQSTTFHSSPGDTETTLLPDDTITSGLVEASTPTHSSTGSLHTTLTPASSTSAGLQEESTTFQSWPSSSDTTPSPPGTTAAPVEVSTTYHSRPSSTPTTHFSASSTTLGRSEESTTVHSSPGATGTALFPTRSATSVLVGEPTTSPISSGSTETTALPGSTTTAGLSEKSTTFYSSPRSPDTTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPGSTTMPGVSQESTASHSSPGSTDTTLSPGSTTASSLGPESTTFHSSPGSTETTLLPDNTTASGLLEASTPVHSSTGSPHTTLSPAGSTTRQGESTTFQSWPSSKDTMPAPPTTTSAFVELSTTSHGSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTAYHSSPGSTQTMHFPESSTASGRSEESRTSHSSTTHTISSPPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSEKSTTFHSSPRSPATTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGPSQESTTSHSSPGSTDTALSPGSTTALSFGQESTTFHSSPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLOGESTAFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTTFYSSPRSPTITLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSPGSTETTVFPRTPTTSVRGEEPTTFHSRPASTHTTLFTEDSTTSGLTEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTTLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGPSQESTTSHSSPGSTDTALSPGSTTALSFGQESTTFHSSPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLQGESTTFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTIFYSSPRSPTTTLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSPGSTETTVFPRSTTTSVRGEEPTTFHSRPASTHTTLFTEDSTTSGLTEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTTLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTSGLSQEPTASHSSQGSTEATLSPGSTTASSLGQQSTTFHSSPGDTETTLLPDDTITSGLVEASTPTHSSTGSLHTTLTPASSTSAGLQEESTTFQSWPSSSDTTPSPPGTTAAPVEVSTTYHSRPSSTPTTHFSASSTTLGRSEESTTVHSSPGATGTALFPTRSATSVLVGEPTTSPISSGSTETTALPGSTTTAGLSEKSTTFYSSPRSPDTTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPGSTTMPGVSQESTASHSSPGSTDTTLSPGSTTASSLGPESTTFHSGPGSTETTLLPDNTTASGLLEASTPVHSSTGSPHTTLSPAGSTTRQGESTTFQSWPNSKDTTPAPPTTTSAFVELSTTSHGSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTTYHSSPGSTQTMHFPESDTTSGRGEESTTSHSSTTHTISSAPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSEKSTTFHSSPRSPATTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGSSQESTTSHSSSGSTDTALSPGSTTALSFGQESTTFHSSPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLQGESTAFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTTFYSSPRSPTTTLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSPGSTETTVFPRSTITSVRREEPTTFHSRPASTHTTLFTEDSTTSGLIEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTKLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTSGLSQEPTTSHSSQGSTEATLSPGSTTASSLGQQSTTFHSSPGDTETTLLPDDTITSGLVEASTPTHSSTGSLHTTLTPASSTSTGLQEESTTFQSWPSSSDTTPSPPSTTAVPVEVSTTYHSRPSSTPTTHFSASSTTLGRSEESTTVHSSPGATGTALFPTRSATSVLVGEPTTSPISSGSTETTALPGSTTTAGLSEKSTTFYSSPRSPDTTLSPASTTSSGVSEESTTSHSRPGSMHTTAFPSSTTMPGVSQESTASHSSPGSTDTTLSPGSTTASSLGPESTTFHSSPGSTETTLLPDNTTASGLLEASTPVHSSTGSPHTTLSPAGSTTRQGESTTFQSWPNSKDTTPAPPTTTSAFVELSTTSHGSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTTYHSSPGSTQTMHFPESNTTSGRGEESTTSHSSTTHTISSAPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSEKSTTFHSSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTAYHSSPGSTQTMHFPESSTASGRSEESRTSHSSTTHTISSPPSTTSALVEEPTSYHSSPGSIATTHFPESSTTSGRSEESTASHSSPDTNGITPLPAHFTTSGRIAESTTFYISPGSMETTLASTATTPGLSAKSTILYSSSRSPDQTLSPASMTSSSISGEPTSLYSQAESTHTTAFPASTTTSGLSQESTTFHSKPGSTETTLSPGSITTSSFAQEFTTPHSQPGSALSTVSPASTTVPGLSEESTTFYSSPGSTETTAFSHSNTMSIHSQQSTPFPDSPGFTHTVLPATLTTTDIGQESTAFHSSSDATGTTPLPARSTASDLVGEPTTFYISPSPTYTTLFPASSSTSGLTEESTTFHTSPSFTSTIVSTESLETLAPGLCQEGQIWNGKQCVCPQGYVGYQCLSPLESFPVETPEKLNATLGMTVKVTYRNFTEKMNDASSQEYQNFSTLFKNRMDVVLKGDNLPQYRGVNIRRLLNGSIVVKNDVILEADYTLEYEELFENLAEIVKAKIMNETRTTLLDPDSCRKAILCYSEEDTFVDSSVTPGFDFQEQCTQKAAEGYTQFYYVDVLDGKLACVNKCTKGTKSQMNCNLGTCQLQRSGPRCLCPNTNTHWYWGETCEFNIAKSLVYGIVGAVMAVLLLALIILIILFSLSQRKRHREQYDVPQEWRKEGTPGIFQKTAIWEDQNLRESRFGLENAYNNFRPTLETVDSGTELHIQRPEMVASTV SEQ ID NO: 41 (hMUC13) MKAIIHLTLLALLSVNTATNQGNSADAVTTTETATSGPTVAAADTTETNFPETASTTANTPSFPTATSPAPPIISTHSSSTIPTPAPPIISTHSSSTIPIPTAADSESTTNVNSLATSDIITASSPNDGLITMVPSETQSNNEMSPTTEDNQSSGPPTGTALLETSTLNSTGPSNPCQDDPCADNSLCVKLHNTSFCLCLEGYYYNSSTCKKGKVFPGKISVTVSETFDPEEKHSMAYQDLHSEITSLFKDVFGTSVYGQTVILTVSTSLSPRSEMRADDKFVNVTIVTILAETTSDNEKTVTEKINKAIRSSSSNFLNYDLTLRCDYYGCNQTADDCLNGLACDCKSDLQRPNPQSPFCVASSLKCPDACNAQHKQCLIKKSGGAPECACVPGYQEDANGNCQKCAFGYSGLDCKDKFQLILTIVGTIAGIVILSMIIALIVTARSNNKTKHIEEENLIDEDFQNLKLRSTGFTNLGAEGSVFPKVRITASRDSQMQNPYSRHSSMPRPDY SEQ ID NO: 42 (hMUC14) MELLQVTILFLLPSICSSNSTGVLEAANNSLVVTTTKPSITTPNTESLQKNVVTPTTGTTPKGTITNELLKMSLMSTATFLTSKDEGLKATTTDVRKNDSIISNVTVTSVTLPNAVSTLQSSKPKTETQSSIKTTEIPGSVLQPDASPSKTGTLTSIPVTIPENTSQSQVIGTEGGKNASTSATSRSYSSIILPVVIALIVITLSVFVLVGLYRMCWKADPGTPENGNDQPQSDKESVKLLTVKTISHESGEHSAQGKTKNSEQ ID NO: 43 (hMUC15) MLALAKILLISTLFYSLLSGSHGKENQDINTTQNIAEVFKTMENKPISLESEANLNSDKENITTSNLKASHSPPLNLPNNSHGITDFSSNSSAEHSLGSLKPTSTISTSPPLIHSFVSKVPWNAPIADEDLLPISAHPNATPALSSENFTwSLVNDTVKTPDNSSITVSILSSEPTSPSVTPLIVEPSGWLTTNSDSFTGFTPYQEKTTLQPTLKFTNNSKLFPNTSDPQKENRNTGIVFGAILGAILGVSLLTLVGYLLCGKRKTDSFSHRRLYDDRNEPVLRLDNAPEPYDVSFGNSSYYNPTLNDSAMPESEENARDGIPMDDIPPLRTSV SEQ ID NO: 44 (MUC16 Homo Sapiens) MLKPSGLPGSSSPTRSLMTGSRSTKATPEMDSGLTGATLSPKTSTGAIVVTEHTLPFTSPDKTLASPTSSVVGRTTQSLGVMSSALPESTSRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGLSSPRTRTSSTEGNFTKEASTYTLTVETTSGPVTEKYTVPTETSTTEGDSTETPWDTRYIPVKITSPMKTFADSTASKENAPVSMTPAETTVTDSHTPGRTNPSFGTLYSSFLDLSPKGTPNSRGETSLELILSTTGYPFSSPEPGSAGHSRiSTSAPLSSSASVLDNKISETSIFSGQSLTSPLSPGVPEARASTMPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQTAETILTFHAFAETMDIPSTHIAKTLASEWLGSPGTLGGTSTSALTTTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESEMTATLVPTLGFTTLDSKIRSPSQVSSSHPTRELRTTGSTSGRQSSSTAAHGSSDILRATTSSTSKASSWTSESTAQQFSEPQHTQWVETSPSMKTERPPASTSVAAPITTSVPSVVSGFTTLKTSSTKGIWLEETSADTLIGESTAGPTTHQFAVPTGISMTGGSSTRGSQGTTHLLTRATASSETSADLTLATNGVPVSVSPAVSKTAAGSSPPGGTKPSYTMVSSVIPETSSLQSSAFREGTSLGLTPLNTRHPFSSPEPDSAGHTKISTSIPLLSSASVLEDKVSATSTFSHHKATSSITTGTPEISTKTKPSSAVLSSMTLSNAATSPERVRNATSPLTHPSPSGEETAGSVLTLSTSAETTDSPNIHPTGTLTSESSESPSTLSLPSVSGVKTTFSSSTPSTHLFTSGEETEETSNPSVSQPETSVSRVRTTLASTSVPTPVFPTMDTWPTRSAQFSSSHLVSELRATSSTSVTNSTGSALPKISHLTGTATMSQTNRDTFNDSAAPQSTTWPETSPRFKTGLPSATTTVSTSATSLSATVMVSKFTSPATSSMEATSIREPSTTILTTETTNGPGSMAVASTNIPIGKGYITEGRLDTSHLPIGTTASSETSMDFTMAKESVSMSVSPSQSMDAAGSSTPGRTSQFVDTFSDDVYHLTSREITIPRDGTSSALTPQMTATHPPSPDPGSARSTWLGILSSSPSSPTPKVTMSSTFSTQRVTTSMIMDTVETSRWNMPNLPSTTSLTPSNIPTSGAIGKSTLVPLDTPSPATSLEASEGGLPTLSTYPESTNTPSIHLGAHASSESPSTIKLTMASVVKPGSYTPLTFPSIETHIHVSTARMAYSSGSSPEMTAPGETNTGSTWDPTTYITTTDPKDTSSAQVSTPHSVRTLRTTENHPKTESATPAAYSGSPKISSSPNLTSPATKAWTITDTTEHSTQLHYTKLAEKSSGFETQSAPGPVSVVIPTSPTIGSSTLELTSDVPGEPLVLAPSEQTTITLPMATWLSTSLTEEMASTDLDISSPSSPMSTFAIFPPMSTPSHELSKSEADTSAIRNTDSTTLDQHLGIRSLGRTGDLTTVPITPLTTTWTSVIEHSTQAQDTLSATMSPTHVTQSLKDQTSIPASASPSHLTEVYPELGTQGRSSSEATTFWKPSTDTLSRElETGPTNIQSTPPMDNITTGSSSSGVTLGIAHLPIGTSSPAETSTNMALERRSSTATVSMAGTMGLLVTSAPGRSISQSLGRVSSVLSESTTEGVTDSSKGSSPRLNTQGNTALSSSLEPSYAEGSQMSTSIPLTSSPTTPDVEFIGGSTFWTKEVTTVMTSDISKSSARTESSSATLMSTALGSTENTGKEKLRTASMDLPSPTPSMEVTPWISLTLSNAPNTTDSLDLSHGVHTSSAGTLATDRSLNTGVTRASRLENGSDTSSKSLSMGNSTHTSMTDTEKSEVSSSIHPRPETSAPGAETTLTSTPGNRAISLTLPFSSIPVEEVISTGITSGPDINSAPMTHSPITPPTIVWTSTGTIEQSTQPLHAVSSEKVSVQTQSTPYVNSVAVSASPTHENSVSSGSSTSSPYSSASLESLDSTISRRNAITSWLWDLTTSLPTTTWPSTSLSEALSSGHSGVSNPSSTTTEFPLFSAASTSAAKQRNPETETHGPQNTAASTLNTDASSVTGLSETPVGASISSEVPLPMAITSRSDVSGLTSESTANPSLGTASSAGTKLTRTISLPTSESLVSFRMNKDPWTVSIPLGSHPTTNTETSIPVNSAGPPGLSTVASDVIDTPSDGAESIPTVSFSPSPDTEVTTISHFPEKTTHSFRTISSLTHELTSRVTPIPGDWMSSAMSTKPTGASPSITLGERRTITSAAPTTSPIVLTASFTETSTVSLDNETTVKTSDILDARKTNELPSDSSSSSDLINTSIASSTMDVTKTASISPTSISGMTASSSPSLFSSDRPQVPTSTTETNTATSPSVSSNTYSLDGGSNVGGTPSTLPPFTITHPVETSSALLAWSRPVRTFSTMVSTDTASGENPTSSNSVVTSVPAPGTWASVGSTTDLPAMGFLKTSPAGEAHSLLASTIEPATAFTPHLSAAVVTGSSATSEASLLTTSESKAIHSSPQTPTTPTSGANWETSATPESLLVVTETSDTTLTSKILVTDTILFSTVSTPPSKFPSTGTLSGASFPTLLPDTPAIPLTATEPTSSLATSFDSTPLVTIASDSLGTVPETTLTMSETSNGDALVLKTVSNPDRSIPGITIQGVTESPLHPSSTSPSKIVAPRNTTYEGSITVALSTLPAGTTGSLVFSQSSENSETTALVDSSAGLERASVMPLTTGSQGMASSGGIRSGSTHSTGTKTFSSLPLTMNPGEVTAMSEITTNRLTATQSTAPKGIPVKPTSAESGLLTPVSASSSPSKAFASLTTAPPSTWGIPQSTLTFEFSEVPSLDTKSASLPTPGQSLNTIPDSDASTASSSLSKSPEKNPRARMMTSTKAISASSFQSTGFTETPEGSASPSMAGHEPRVPTSGTGDPRYASESMSYPDPSKASSAMTSTSLASKLTTLFSTGQAARSGSSSSPISLSTEKETSFLSPTASTSRKTSLFLGPSMARQPNILVHLQTSALTLSPTSTLNMSQEEPPELTSSQTIAEEEGTTAETQTLTFTPSETPTSLLPVSSPTEPTARRKSSPETWASSISVPAKTSLVETTDGTLVTTIKMSSQAAQGNSTWPAPAEETGTSPAGTSPGSPEVSTTLKIMSSKEPSISPEIRSTVRNSPWKTPETTVPMETTVEPVTLQSTALGSGSTSISHLPTGTTSPTKSPTENMLATERVSLSPSPPEAWTNLYSGTPGGTRQSLATMSSVSLESPTARSITGTGQQSSPELVSKTTGMEFSMWHGSTGGTTGDTHVSLSTSSNILEDPVTSPNSVSSLTDKSKHKTETWVSTTAIPSTVLNNKIMAAEQQTSRSVDEAYSSTSSWSDQTSGSDITLGASPDVTNTLYITSTAQTTSLVSLPSGDQGITSLTNPSGGKTSSASSVTSPSIGLETLRANVSAVKSDIAPTAGHLSQTSSPAEVSILDVTTAPTPGISTTITTMGTNSISTTTPNPEVGMSTMDSTPATERRTTSTEHPSTWSSTAASDSWTVTDMTSNLKVARSPGTISTMHTTSFLASSTELDSMSTPHGRITVIGTSLVTPSSDASAVKTETSTSERTLSPSDTTASTPISTFSRVQRMSISVPDILSTSWTPSSTEAEDVPVSMVSTDHASTKTDPNTPLSTFLFDSLSTLDWDTGRSLSSATATTSAPQGATTPQELTLETMISPATSQLPFSIGHITSAVTPAAMARSSGVTFSRPDPTSKKAEQTSTQLPTTTSAHPGQVPRSAATTLDVIPHTAKTPDATFQRQGQTALTTEARATSDSWNEKEKSTPSAPWITEMMNSVSEDTIKEVTSSSSVLKDPEYAGHKLGIWDDFlPKFGKAAHMRELPLLSPPQDKEAIHPSTNTVETTGWVTSSEHASHSTIPAHSASSKLTSPVVTTSTREQAIVSMSTTTWPESTRARTEPNSFLTIELRDVSPYMDTSSTTQTSIISSPGSTAITKGPRTEITSSKRISSSFLAQSMRSSDSPSEAITRLSNFPAMTESGGMILAMQTSPPGATSLSAPTLDTSATASWTGTPLATTQRFTYSEKTTLFSKGPEDTSQPSPPSVEETSSSSSLVPIHATTSPSNILLTSQGHSPSSTPPVTSVFLSETSGLGKTTDMSRISLEPGTSLPPNLSSTAGEALSTYEASRDTKAIHHSADTAVTNMEATSSEYSPIPGHTKPSKATSPLVTSHIMGDITSSTSVFGSSETTEIETVSSVNQGLQERSTSQVASSATETSTVITHVSSGDATTHVTKTQATFSSGTSISSPHQFITSTNTFTDVSTNPSTSLIMTESSGVTITTQTGPTGAATQGPYLLDTSTMPYLTETPLAVTPDFMQSEKTTLISKGPKDVTWTSPPSVAETSYPSSLTPFLVTTIPPATSTLQGQHTSSPVSATSVLTSGLVKTTDMLNTSMEPVTNSPQNLNNPSNElLATLAATTDIETIHPSINKAVTNMGTASSAHVLHSTLPVSSEPSTATSPMVPASSMGDALASISIPGSETTDIEGEPTSSLTAGRKENSTLQEMNSTTESNIILSNVSVGAITEATKMEVPSFDATFIPTPAQSTKFPDIFSVASSRLSNSPPMTISTHMTTTQTGSSGATSKIPLALDTSTLETSAGTPSVVTEGFAHSKITTAMNNDVKDVSQTNPPFQDEASSPSSQAPVLVTTLPSSVAFTPQWHSTSSPVSMSSVLTSSLVKTAGKVDTSLETVTSSPQSMSNTLDDISVTSAATTDIETTHPSINTVVTNVGTTGSAFESHSTVSAYPEPSKVTSPNVTTSTMEDTTISRSIPKSSKTTRTETETTSSLTPKLRETSISQEITSSTETSTVPYKELTGATTEVSRTDVTSSSSTSFPGPDQSTVSLDISTETNTRLSTSPIMTESAEITITTQTGPHGATSQDTFTMDPSNTTPQAGIHSAMTHGFSQLDVTTLMSRIPQDVSWTSPPSVDKTSSPSSFLSSPAMTTPSLISSTLPEDKLSSPMTSLLTSGLVKITDILRTRLEPVTSSLPNFSSTSDKILATSKDSKDTKEIFPSINTEETNVKANNSGHESHSPALADSETPKATTQMVITTTVGDPAPSTSMPVHGSSETTNIKREPTYFLTPRLRETSTSQESSFPTDTSFLLSKVPTGTITEVSSTGVNSSSKISTPDHDKSTVPPDTFTGEIPRVFTSSIKTKSAEMTITTQASPPESASHSTLPLDTSTTLSQGGTHSTVTQGFPYSEVTTLMGMGPGNVSWMTTPPVEETSSVSSLMSSPAMTSPSPVSSTSPQSIPSSPLPVTALPTSVLVTTTDVLGTTSPESVTSSPPNLSSITHERPATYKDTAHTEAAMHHSTNTAVTNVGTSGSGHKSQSSVLADSETSKATPLMSTTSTLGDTSVSTSTPNISQTNQIQTEPTASLSPRLRESSTSEKTSSTTETNTAFSYVPTGAITQASRTEISSSRTSISDLDRPTIAPDISTGMITRLFTSPIMTKSAEMTVTTQTTTPGATSQGILPWDTSTTLFQGGTHSTVSQGFPHSEITTLRSRTPGDVSWMTTPPVEETSSGFSLMSPSMTSPSPVSSTSPESIPSSPLPVTALLTSVLVTTINVLGTTSPETVTSSPPNLSSPTQERLTTYKDTAHTEAMHASMHTNTAVANVGTSISGHESQSSVPADSHTSKATSPMGITFAMGDTSVSTSTPAFFETRIQTESTSSLIPGLRDTRTSEEINTVTETSTVLSEVPTTTTTEVSRTEVITSSRTTISGPDHSKMSPYISTETITRLSTFPFVTGSTEMAITNQTGPIGTISQATLTLDTSSTASWEGTHSPVTQRFPHSEETTTMSRSTKGVSWQSPPSVEETSSPSSPVPLPAITSHSSLYSAVSGSSPTSALPVTSLLTSGRRKTIDMLDTHSELVTSSLPSASSFSGEILTSEASTNTETIHFSENTAETNMGTTNSMHKLHSSVSIHSQPSGHTPPKVTGSMMEDAIVSTSTPGSPETKNVDRDSTSPLTPELKEDSTALVMNSTTESNTVFSSVSLDAATEVSRAEVTYYDPTFMPASAQSTKSPDISPEASSSHSNSPPLTISTHKTIATQTGPSGVTSLGQLTLDTSTIATSAGTPSARTQDFVDSETTSVMNNDLNDVLKTSPFSAEEANSLSSQAPLLVTTSPSPVTSTLQEHSTSSLVSVTSVPTPTLAKITDMDTNLEPVTRSPQNLRNTLATSEATTDTHTMHPSINTAMANVGTTSSPNEFYFTVSPDSDPYKATSAVVITSTSGDSIVSTSMPRSSAMKKIESETTFSLIFRLRETSTSQKIGSSSDTSTVFDKAFTAATTEVSRTELTSSSRTSIQGTEKPTMSPDTSTRSVTMLSTFAGLTKSEERTIATQTGPHRATSQGTLTWDTSITTSQAGTHSAMTHGFSQLDLSTLTSRVPEYISGTSPPSVEKTSSSSSLLSLPAITSPSPVPTTLPESRPSSPVHLTSLPTSGLVKTTDMLASVASLPPNLGSTSHKIPTTSEDIKDTEKMYPSTNIAVTNVGTTTSEKESYSSVPAYSEPPKVTSPMVTSFNIRDTIVSTSMPGSSEITRIEMESTFSVAHGLKGTSTSQDPIVSTEKSAVLHKLTTGATETSRTEVASSRRTSIPGPDHSTESPDISTEVIPSLPISLGITESSNMTIITRTGPPLGSTSQGTFTLDTPTTSSRAGTHSMATQEFPHSEMTTVMNKDPEILSWTIPPSIEKTSFSSSLMPSPAMTSPPVSSTLPKTIHTTPSPMTSLLTPSLVMTTDTLGTSPEPTTSSPPNLSSTSHVILTTDEDTTAIEAMHPSTSTAATNVETTCSGHGSQSSVLTDSEKTKATAPMDTTSTMGHTTVSTSMSVSSETTKIKRESTYSLTPGLRETSISQNASFSTDTSIVLSEVPTGTTAEVSRTEVTSSGRTSIPGPSQSTVLPEISTRTMTRLFASPTMTESAEMTIPTQTGPSGSTSQDTLTLDTSTTKSQAKTHSTLTQRFPHSEMTTLMSFIGPGDMSWQSSPSLENPSSLPSLLSLPATTSPPPISSTLPVTISSSPLPVTSLLTSSPVITTDMLHTSPELVTSSPPKLSHTSDERLTTGKDTTNTEAVHPSTNTAASNVEIPSFGHESPSSALADSETSKATSPMFITSTQEDTTVAISTPHFLETSRIQKESISSLSPKLRETGSSVETSSAIETSAVLSEVSIGATTEISRTEVISSSRTSISGSAESTMLPEISTTRKIIKFPTSPILAESSEMTIKTQTSPPGSTSESTFTLDTSTTPSLVITHSTMTQRLPHSEITTLVSRGAGDVPRPSSLPVEETSPPSSQLSLSAMISPSPVSSTLPASSHSSSASVTSPLTPGQVKTTEVLDASAEPETSSPPSLSSTSVEILATSEVTTDTEKIHPFPNTAVTKVGTSSSGHESPSSVLPDSETTKATSAMGTISIMGDTSVSTLTPALSNTRKIQSEPASSLTTRLRETSTSEETSLATEANTVLSKVSTGATTEVSRTEAISFSRTSMSGPEQSTMSQDISIGTIPRISASSVLTESAKMTITTQTGPSESTLESTLNLNTATTPSWVETHSIVIQGFPHPEMTTSMGRGPGGVSWPSPPFVKETSPPSSPLSLPAVTSPHPVSTTFLAHIPPSPLPVTSLLTSGPATTTDILGTSTEPGTSSSSSLSTTSHERLTTYKDTAHTEAVHPSTNTGGTNVATTSSGYKSQSSVLADSSPMCTTSTMGDTSVLTSTPAFLETRRIQTELASSLTPGLRESSGSEGTSSGTKMSTVLSKVPTGATTEISKEDVTSIPGPAQSTISPDISTRTVSWFSTSPVMTESAEITMNTHTSPLGATTQGTSTLATSSTTSLTMTHSTISQGFSHSQMSTLMRRGPEDVSWMSPPLLEKTRPSFSLMSSPATTSPSPVSSTLPESISSSPLPVTSLLTSGLAKTTDMLHKSSEPVTNSPANLSSTSVEILATSEVTTDTEKTHPSSNRIVTDVGISSSGHESTSFVLADSQTSKVTSPMVITSTMEDTSVSTSTPGFFETSRIQTEPTSSLTLGLRKTSSSEGTSLATEMSTVLSGVPTGATAEVSRTEVTSSSRTSISGFAQLTVSPETSTETITRLPTSSIMTESAEMMIKTQTDPPGSTPESTHTVDISTTPNWVETHSTVTQRFSHSEMTTLVSRSPGDMLWPSQSSVEETSSASSLLSLPATTSPSPVSSTLVEDFPSASLPVTSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEDMQPSTHTAVTNVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDISTEAITRLSTSPIMTESAESAITIETGSPGATSEGTLILDTSITTFwSGTHSTASPGFSHSEMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSALPESISSSPHPVTALLTLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREISTSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRISTPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKTTDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYSSYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISSEVITRLSTSPIKAESTEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGDVPWLSHPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSEPETSSPPNLSSTSAEILATTEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPTGATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPSGATSQGTFTLDSSSTASWPGTHSATTQRFPQSVVTTPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNITSDESLATSKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSSIRDNMVSTTMPGSSGITRIElESMSSLTPGLRETRTSQDITSSTETSTVLYKMSSGATPEVSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQVTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIPATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVDDTTVFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLYGVPTSATTEVSMTEIMSSNRTHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLATTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSPFVEKTSSSSSLLSLPVTTSPSVSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSGFRKTNMSLDTSSVIPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDWPPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETISTGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSSSTFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSTPILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQTSTSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASRQMASTILTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVLNRESETTASLVSRSGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGADVSSAlPTNISPSELDALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETSSTIPRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPTLTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNSPGEPATTVSLVTHPAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEVPGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVVTSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVASSRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNSTSIFTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIPILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTSLVTSSRAVTSTTIPTLTISSDEPETTTSLVTHSEAKMISAIPTLAVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPSTVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPESPHESEATASWVTHPAVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSGTDTSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPGASKMLTSLVISSGTDSTTTFPTLTETPYEPETTAIQLIHPAETNTMVPKTTPKFSHSKSDTTLPVAITSPGPEASSAVSTTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTVTWLTHPAETSTTVSGTIPNFSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPGEPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPDATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALLSTHPRTGTSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDLSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPPKTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLASESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMVPFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSAMAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTAAGPLLMPFTLNFTITNLQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTAAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKHEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKHEAATGVDTICTHRVDPIGPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLDREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPTTASPLLVLFTINCTITNLQYEEDMRRIGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPKKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLRPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAACTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRVSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGHTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSAMAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRRFNTTERVLQGLLTPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITDLHYEENMQHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSVTELGPYTLDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEEDMRHPGSRKFSTTERVLQGLLKPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTTLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTQDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPAXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICTHAVOPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVXXGTSGTPSSXPXXTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGATTGMDAICTHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSAMAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLIPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTQDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETTGTPSSFPGHTEPGPLLIPFTFNFTITNLRYEENMQHPGSRKFNTTERVLQGLLTPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVDGFNPWSSVPTTSTPGTSTVHLATSGTPSPLPGHTAPVPLLIPFTLNFTITDLHYEENMQHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPITSTPGTSTVHLATSGTPSSLPGHTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLHGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTNSITELGPYTLDRDSLYVNGFTHRSSMPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKRGAATGVDTICTHRLDPLNPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFHPRSSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTITNLHYEENMQHPGSRKFNTTERVLQGLLTPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHRSSVPTTSSPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHRTSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYCELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHWSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLQGLLSPIFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHRSFGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLTPLFRNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHQTFAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATRVDAVCTHRPDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSVTELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEVDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGFTHRNFVPITSTPGTSTVHLGTSETPSSLPRPIVPGPLLVPFTLNFTITNLQYEEAMRHPGSRKFNTTERVLQGLLRPLFKNTSIGPLYSSCRLTLLRPEKDKAATRVDAICTHHPDPQSPGLNREQLYWELSQLTHGITELGPYTLDRDSLYVDGFTHWSPIPTTSTPGTSIVNLGTSGIPPSLPETTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLVLFTLNFTITNLRYEENMQHPGSRKFNITERVLQGLLRSLFKSTSVGPLYSGCRLILLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGHYALDNDSLFVNGFTHRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGSRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMKHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGLDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQDKVITLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGLITCLICGVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ SEQ ID NO: 45 (hMUC17) MPRPGTMALCLLTLVLSLLPPQAAAEQDLSVNRAVWDGGGCISQGDVLNRQCQQLSQHVRTGSAANTATGTTSTNVVEPRMYLSCSTNPEMTSIESSVTSDTPGVSSTRMTPTESRTTSESTSDSTTLFPSSTEDTSSPTTPEGTDVPMSTPSEESISSTMAFVSTAPLPSFEAYTSLTYKVDMSTPLTTSTQASSSPTTPESTTIPKSTNSEGSTPLTSMPASTMKVASSEAITLLTTPVEISTPVTISAQASSSPTTAEGPSLSNSAPSGGSTPLTRMPLSVMLVVSSEASTLSTTPAATNIPVITSTEASSSPTTAEGTSIPTSTYTEGSTPLTSTPASTMPVATSEMSTLSITPVDTSTLVTTSTEPSSLPTTAEATSMLTSTLSEGSTPLTNMPVSTILVASSEASTTSTIPVDSKTFVTTASEASSSPTTAEDTSIATSTPSEGSTPLTSMPVSTTPVASSEASNLSTTPVDSKTQVTTSTEASSSPPTAEVNSMPTSTPSEGSTPLTSMSVSTMPVASSEASTLSTTPVDTSTPVTTSSEASSSSTTPEGTSIPTSTPSEGSTPLTNMPVSTRLVVSSEASTTSTTPADSNTFVTTSSEASSSSTTAEGTSMPTSTYSERGTTITSMSVSTTLVASSEASTLSTTPVDSNTPVTTSTEATSSSTTAEGTSMPTSTYTEGSTPLTSMPVNTTLVASSEASTLSTTPVDTSTPVTTSTEASSSPTTADGASMPTSTPSEGSTPLTSMPVSKTLLTSSEASTLSTTPLDTSTHITTSTEASCSPTTTEGTSMPISTPSEGSPLLTSIPVSITPVTSPEASTLSTTPVDSNSPVTTSTEVSSSPTPAEGTSMPTSTYSEGRIPLTSMPVSTTLVATSAISTLSTTPVDTSTPVTNSTEARSSPTTSEGTSMPTSTPGEGSTPLTSMPDSTTPVVSSEARTLSATPVDTSTPVTTSTEATSSPTTAEGTSIPTSTPSEGTTPLTSTPVSHTLVANSEASTLSTTPVDSNTPLTTSTEASSPPPTAEGTSMPTSTPSEGSTPLTRMPVSTTMVASSETSTLSTTPADTSTPVTTYSQASSSSTTADGTSMPTSTYSEGSTPLTSVPVSTRLVVSSEASTLSTTPVDTSIPVTTSTEASSSPTTAEGTSIPTSPPSEGTTPLASMPVSTTLVVSSEANTLSTTPVDSKTQVATSTEASSPPPTAEVTSMPTSTPGERSTPLTSMPVRHTPVASSEASTLSTSPVDTSTPVTTSAETSSSPTTAEGTSLPTSTTSEGSTLLTSIPVSTTLVTSPEASTLLTTPVDTKGPVVTSNEVSSSPTPAEGTSMPTSTYSEGRTPLTSIPVNTTLVASSAISILSTTPVDNSTPVTTSTEACSSPTTSEGTSMPNSNPSEGTTPLTSIPVSTTPVVSSEASTLSATPVDTSTPGTTSAEATSSPTTAEGISIPTSTPSEGKTPLKSIPVSNTPVANSEASTLSTTPVDSNSPVVTSTAVSSSPTPAEGTSIAISTPSEGSTALTSIPVSTTTVASSElNSLSTTPAVTSTPVTTYSQASSSPTTADGTSMQTSTYSEGSTPLTSLPVSTMLVVSSEANTLSTTPIDSKTQVTASTEASSSTTAEGSSMTISTPSEGSPLLTSIPVSTTPVASPEASTLSTTPVDSNSPVITSTEVSSSPTPAEGTSMPTSTYTEGRTPLTSITVRTTPVASSAISTLSTTPVDNSTPVTTSTEARSSPTTSEGTSMPNSTPSEGTTPLTSIPVSTTPVLSSEASTLSATPIDTSTPVTTSTEATSSPTTAEGTSIPTSTLSEGMTPLTSTPVSHTLVANSEASTLSTTPVDSNSPVVTSTAVSSSPTPAEGTSIATSTPSEGSTALTSIPVSTTTVASSETNTLSTTPAVTSTPVTTYAQVSSSPTTADGSSMPTSTPREGRPPLTSIPVSTTTVASSElNTLSTTLADTRTPVTTYSQASSSPTTADGTSMPTPAYSEGSTPLTSMPLSTTLVVSSEASTLSTTPVDTSTPATTSTEGSSSPTTAGGTSIQTSTPSERTTPLAGMPVSTTLVVSSEGNTLSTTPVDSKTQVINSTEASSSATAEGSSMTISAPSEGSPLLTSIPLSTTPVASPEASTLSTTPVDSNSPVITSTEVSSSPIPTEGTSMQTSTYSDRRTPLTSMPVSTTVVASSAISTLSTTPVDTSTPVTNSTEARSSPTTSEGTSMPTSTPSEGSTPFTSMPVSTMPVVTSEASTLSATPVDTSTPVTTSTEATSSPTTAEGTSIPTSTLSEGTTPLTSIPVSHTLVANSEVSTLSTTPVDSNTPFTTSTEASSPPPTAEGTSMPTSTSSEGNTPLTRMPVSTTMVASFETSTLSTTPADTSTPVTTYSQAGSSPTTADDTSMPTSTYSEGSTPLTSVPVSTMPVVSSEASTHSTTPVDTSTPVTTSTEASSSPTTAEGTSIPTSPPSEGTTPLASMPVSTTPVVSSEAGTLSTTPVDTSTPMTTSTEASSSPTTAEDIVVPISTASEGSTLLTSIPVSTTPVASPEASTLSTTPVDSNSPVVTSTEISSSATSAEGTSMPTSTYSEGSTPLRSMPVSTKPLASSEASTLSTTPVDTSIPVTTSTETSSSPTTAKDTSMPISTPSEVSTSLTSILVSTMPVASSEASTLSTTPVDTRTLVITSTGTSSSPTTAEGSSMPTSTPGERSTPLTNILVSTTLLANSEASTLSTTPVDTSTPVTTSAEASSSPTTAEGTSMRISTPSDGSTPLTSILVSTLPVASSEASTVSTTAVDTSIPVTTSTEASSSPTTAEVTSMPTSTPSETSTPLTSMPVNHTPVASSEAGTLSTTPVDTSTPVTTSTKASSSPTTAEGIVVPISTASEGSTLLTSIPVSTTPVASSEASTLSTTPVDTSIPVTTSTEGSSSPTTAEGTSMPISTPSEVSTPLTSILVSTVPVAGSEASTLSTTPVDTRTPVTTSAEASSSPTTAEGTSMPISTPGERRTPLTSMSVSTMPVASSEASTLSRTPADTSTPVTTSTEASSSPTTAEGTGIPISTPSEGSTPLTSIPVSTTPVAIPEASTLSTTPVDSNSPVVTSTEVSSSPTPAEGTSMPISTYSEGSTPLTGVPVSTTPVTSSAlSTLSTTPVDTSTPVTTSTEAHSSPTTSEGTSMPTSTPSEGSTPLTYMPVSTMLVVSSEDSTLSATPVDTSTPVTTSTEATSSTTAEGTSIPTSTPSEGMTPLTSVPVSNTPVASSEASILSTTPVDSNTPLTTSTEASSSPPTAEGTSMPTSTPSEGSTPLTSMPVSTTTVASSETSTLSTTPADTSTPVTTYSQASSSPPIADGTSMPTSTYSEGSTPLTNMSFSTTPVVSSEASTLSTTPVDTSTPVTTSTEASLSPTTAEGTSIPTSSPSEGTTPLASMPVSTTPVVSSEVNTLSTTPVDSNTLVTTSTEASSSPTIAEGTSLPTSTTSEGSTPLSIMPLSTTPVASSEASTLSTTPVDTSTPVTTSSPTNSSPTTAEVTSMPTSTAGEGSTPLTNMPVSTTPVASSEASTLSTTPVDSNTFVTSSSQASSSPATLQVTTMRMSTPSEGSSSLTTMLLSSTYVTSSEASTPSTPSVDRSTPVTTSTQSNSTPTPPEVITLPMSTPSEVSTPLTIMPVSTTSVTISEAGTASTLPVDTSTPVITSTQVSSSPVTPEGTTMPIWTPSEGSTPLTTMPVSTTRVTSSEGSTLSTPSVVTSTPVTTSTEAISSSATLDSTTMSVSMPMEISTLGTTILVSTTPVTRFPESSTPSIPSVYTSMSMTTASEGSSSPTTLEGTTTMPMSTTSERSTLLTTVLISPISVMSPSEASTLSTPPGDTSTPLLTSTKAGSFSIPAEVTTIRISITSERSTPLTTLLVSTTLPTSFPGASIASTPPLDTSTTFTPSTDTASTPTIPVATTISVSVITEGSTPGTTIFIPSTPVTSSTADVFPATTGAVSTPVITSTELNTPSTSSSSTTTSFSTTKEFTTPAMTTAAPLTYVTMSTAPSTPRTTSRGCTTSASTLSATSTPHTSTSVTTRPVTPSSESSRPSTITSHTIPPTFPPAHSSTPPTTSASSTTVNPEAVTTMTTRTKPSTRTTSFPTVTTTAVPTNTTIKSNPTSTPTVPRTTTCFGDGCQNTASRCKNGGTWDGLKCQCPNLYYGELCEEVVSSIDIGPPETISAQMELTVTVTSVKFTEELKNHSSQEFQEFKQTFTEQMNIVYSGIPEYVGVNITKLRLGSVVVEHDVLLRTKYTPEYKTVLDNATEVVKEKITKVITQQIMINDICSDMMCFNTTGTQVQNITVTQYDPEEDCRKMAKEYGDYFVVEYRDQKPYCISPCEPGFSVSKNCNLGKCQMSLSGPQCLCVTTETHWYSGETCNQGTQKSLVYGLVGAGVVLMLIILVALLMLVFRSKREVKRQKYRLSQLYKWQEEDSGPAPGTFQNIGFDICQDDDSIHLESIYSNFQPSLRHIDPETKIRIQRPQVMTTSFSEQ ID NO: 46 (hMUC19) XXXXXXXXXXSGSTGVSAGSITASPGASATSSESSKSGSTEGSVEATTSAGSGNTAGTSGTGDTGPGNTAGATGSSTGQTDTSGPSAKVTGNYGQSSEIPGTIKSSSDVSGTMGQSDTTSGPSVAVTRTSEQSSGVTVASEPSVGVSGTTGPLAEISGTTRPLVSGLRTTGSSAEGSGTTGPSSRESVTTRPLAEGSGTSGQSVTGSRATGLSATELGTTVSFTGGLGTSRSSARETRTTGPSADGSGTTGPSVVRSGTTRLSVGVTRATESSPGVTGTTTPSAEESRTTGPSVLVTGTTGQSGQGSGTTGKSFIESGPSVVGSGTTGPTSAGLGTTAPSTRRSSTTKPSVGRTGTTGQSGAESGTTEPSARVAGVTGTSAEVSGRIEPSATESSTSRPLGETTGTTIPSMEGSEATGPSVIGSETTRLSVIGSGTTGTSSGGSGATRSSGGGMGTTGQSTARSETTGPLFGLTGTFGQSATVTGTSSNSAGVTTPEKSPGVAMTTGLLVEGSATTQPRILESETTESSAGVIVTSGQSARVTGATGPSAGETGTTEPSTEGSVAAVLFVIGSETTRPLDIGSGTTGTLSGGSSTTRSSDGTTGTTRKSTARSETTGLSGLTGTSGQLAGVTGTSSKSAGVTVTSEKSAGVAVITGSFVERPVTTGPPLLESETTRPSGGVTVTSGQSARVTETVGASAGVTGTTGPSTEGSGATGPSVVGSGTTRPLAGESGTTESSAGVTGTRPSSSRESATTGPSDEGSGTTGLSAGVTVTSGQSVRKTGTTGAPAGVTETTRPSVVKSGTTGPSVIGTRTTGTSSGGSGATRSSGGETETTGQSAVKSGTTESFTRLTRTSGQSAGMTGTSAQSAGVALTSPFVEGLVTTGSSTVGLETTRPSAVGSGKTGPPVVKAQTTGPSAGVTVTSGQSARMTGASGPSVGVTGTTGPASKGLGTIRPSVVGLETTELSAEGSGTTGPPIVGETTVPSAGVTVTSGYSDRVTGATEPLAGVTGTIKPSVAGSVTTGPSVTGVETTAKTTSGGLSTTISSVGGTGTTGQSPERSGTTGPFTGLTGTSAQSAGVTMTSIQSAGVLVTTGLNVDGLGTTGKALIGSGTTGLSAEATGTIGPSTEGLEKTGPSITGSGTTRPLVTESWTAGTSSGGHSTTSPSVRGTETTGQSAAESVTTGPVTGYTETSGPSAGVTVTPRQSPTVTQTTGSSAAVSGTTVQSLTVSGTTRPSSGQTEITGSSVKESGTTESSAVRSGTTGPTAGVTGTNGPSSAGVTGITGSSPGVTGTTGSSPGVTGTTGSSARSGTSIPSVGKTGTTRTSVEESRTTRPSAGITGTNGLSAEVTGTTGPLAGVTGTTGPSAGVTRTTGLSAGETGTTGLSPGVTRTTRSSAGLTGKTGLSAGVTGKTGLSAEVTGTTRLSAGVTGTTGPSPGVTGTTGTPAGVTGTTELSAGVTGKTGLSSEVTETTGLSYGVKRTIGLSAGSTGTSGQSAGVAGTTTLSAEVTGTTRPSAGVTGTTGLSAEVTEITGISAVVTGTTGPSAGVTETTGSSAGVAGTTRLSAGVTGITGLSAGVTGTTGLSTEVTGTTGPSAGATGTTGLSVGVTGITGLSDVVTETTGSSARSGTGIPSVGETRTTSTSVEESRTTRPSAGIMGTNGLPAEVTGTTEPLAGGTGTTGILAGVTGTTGLSAGETGKIGSSAGVTGKTGSSARVTGKTGPSAEVTGKTGLSAGVTGTTGLSPGVTGTSGLSAEVTGTTGPSAEATGLPGVSAGVTGTTGSLAGGTGTIGLSAGVTGTTGSSAGVTGTTGLSAGVTGIAGLSAGVTGITGPSAGVTGTTTVSAGVTGTTGLSAEATEITGLSAGVTGTTGLSAGVTETIRLSAGVTGTIRSSAGVTGITGLSAGVTGTTGPSAGVTGSTGLLAGVTETTGQSAKVTGTTGQSVGVTGTTRSSGGVTGITGLSAGVTGTNGLSAVTGMTGLSAEVTGTTGLSVGVTGIAGLSAGVTGITGPSAGITGTTTISAGVTGTSGLSAEATGITGLSAGVTGKTGLSAGVTETIGLSAEATGTIGSSPGVTGTTGSSTGVTGITGLSAGVTGTTGLSTEVTGTTGPSAGVTRTTGLSAGVTGITGLSAIVTETTGSSARSGTSIPSVGETGTTRTSVEESRTTRPSAGITGTTGLSAGVTGTVGSSAVVTGTTGLSAGVTGTTGPSAEETGATGPSAEVTETTGPSAGVTGTGRLSAEVTGTTGPSAEVTGLPGESAEVTGTIGSPAGVTGTTQLSAVVTGITGLSAEVTGTTGLSAGVTGITGLSAEVTRTTGLSAGVTGTIGLSAGVTGTTRPSAGVTGTTGQSAEVTGTTEPSAGLTETTGSSTGVTGATGPLAGVTGTTGISTEVTGTTGPSARVTGTTVLSAGVTGITGLSAIVTETTGSSARSGTSTPSVGETGTTRTSVEESRATRPSAGITGTNGQSAEVTWITGPLAGVTGTTGISAGVTGTTGLSAGVTGTIGSSAVVTGINGLSAGVTGTTGPSAEETGATGPSAEVTGTTGPSAEETGATGPSAEVTGTTGPSGGVTGTNGLSAEVTGTTGPSAEVTGLPGVSAGVTGTIGSPAAVTGTIRPSAVVTGITGLSAEVTGTTGLSAWVTGIAGLSAGVTETIGSSAGVTGTNGLSAEATGTTGPSAGVTGTTGLSAGVTGTAGLSARVIESTGLSAGVTGTTGLSAGVTGTTGPSAGITGTNGLSAEVTGTTGPLAGVTGTIGLSAGVTGIAGLSAGVTESTGLSAGVTGTIRSSAVVTGINGLSAGVTGTTGPSAEETGATGPSAEVTGTTGPSGGVTGTSGISAEVTGTTGPSAEVTGLPGVSAGVTGTIGSPAAVTGTTRPSAVVTGISGLSAEVTGTTGLSAGVTETIGSSAGVTGTNGLSAEATETTGPSAGVTGTTGLSAGVTGTTGPSAGIAGTNGLSAGVTGTTGLSARVTESTGLSAGVTGTIGSSAVVTETTRLSSGVTGTIGPSAEETGATGLSAEVTGTTGSLAEVTGTTGLSAGVTGTIGSSAVVTGTTGLSAGITGTNGLSAEVTGTAGPLAGVTGTTGLSAGVTGTTGLSAGVTETTGQSAGVTESTGLSPGVTGTIGSSAVVTGIKGLSAGVTGTTGPSAEETGATGPSAEVTGTTGPSGGVTGTSVLSVEVTGTTGPSAEVTGLPGVSAGLTGTIGSPAAVRGTTWPSAVVTGISGLSGEVTGTTGLSAGVTGIGGLSAGVTGTIGSSAGVTGTNALSAEATGTTGPSAGVTGTTGLSAGVTGTTGLSAGVIGTIRSSAVVTETTGLSAGVTGTTGPSAGIAGTNGLSAEVTGTTGLSAGMTGTTGLSARVTESTGLSAGVTGTIGSSAVVTETTRLSAGVTGTIGPSAEETGATGLSAEVTRTTGSLAGVIGTTGPSAVVIGKTELSAEVTGTTELSAEVTEKTGPSAEVTGKTGLSAGVMETTGPSAEVTGTTGSSAGVTGTTGPSAGVTGTTGPSAEATGLPGVSAGVTGTIGSPAGVTGTARLSAVVTGISGLSAEVTGTTGLSTGVTGIAGHSAAVTGITRPSAGVTGTTIVSAGVTGTIGLSAEATGITLPSAGVTETTGLSAGVTETIGLSAGVTGTIGSSAGVTEITGLSAGVTGTTGPSAGVTGSTVLSAGVTATTGQSVGVTGTTGPSAGVTGTTGLSAGVTGIAGLSAGVTGITGPSAGVTGTTTVSAGVTGTTGLSAEATEITGLSAGVTGTTGLSAGVTGIAGLSAGVTETIGSSAGVTGTNGLSAEATGKTGPSAGVTGTTGLSAGVTGTTGLSAGVTETIGLSAGVTGTIGSSAGVKGTTGQSAEVTGATGQSVGVTGTTRSSGGVTGITGLSAGLRGTTVSSAKAGTSIPLTGKTGTTRTSVEESTTTGPSAGITGTNGLSAEMTGTNELSAGVTGTIGSSAGVTGTTGLSVEATVTTGLSAGVTGTTVPLAGVTWTPGPSAGVTGIAALSAGVTGKSGLSAGVTGKTGLSAGVTGTTGPSAEATGKTGLSAGVTGITGPFAEVTGTTGLSAGVIGTTGSSAEVTGITGLSAGVTGKTRSSAGVTGTTGLSAKSGTSIPSAGKTGTTKTSVEESRTTRPSAGITGTNGLPARVTGTXXXXXXXXXXGTSGVAPGTTVAPGSFSTAATTSPGASGVTGTGPTAETTTFLGGSSTTGAEIKSGATTGAPGSKTGTAKVLSGTTVASGSSNSEATTFSGITEAVTVPSKNGSMTTALGSQLSSSQTVIPGSSGTISHTTVAPGSSVTGTTTGASDDQVTGSKTGTTGVALSTTVAPGSSSTEATTSTGVHRTTVVGQKTGATTRGSAKQGTRSTIEATTSFRGTGTTGSGMNTGTTGVVSGNTISPSSFNTEATSGTSERPNPGSEIGTTGIVSGTTVAPGSSNTEATTSLGNGGTTEAGSKIVTTGITTGTTIVPGSFNTKATTSTDVGVATGVGMATGITNIISGRSQPTGSKTGYTVTGSGTTALPGGFRTGNTPGSTGVTSSQEGTTVVSSGITGIPETSISGPSKEASDKTTAPGPPTTVTASTGVKETSETGVQTGSTLVTAGVPTRPQVSQPETTVVATREVETENKTECLASLPPAPVCHGPLGEEKSPGDIWTANCHRGICTDAKTIDCKPEECPSPPTCKTGEKLVKFQSNDTCCEIGYCEPRTCLFNNTDYEIGASFDDPSNPCVSYSCKDTGFAAVVQDCPKQTWCAEANRIYDSKKCCYTCKNNCRSSLVNVTVIYSGCKKRVQMAKCTGECEKTAKYNHDILLLEHSCLCCREENYELRDIVLDCPDGSTIPYQYKHITTCSCLDICQLYTTFMYSSEQ ID NO: 47 (hMUC20) MGCLWGLALPLFFFCWEVGVSGSSAGPSTRRADTAMTTDDTEVPAMTLAPGHAALETQTLSAETSSRASTPAGPIPEAETRGAKRISPARETRSFTKTSPNFMVLIATSVETSAASGSPEGAGMTTVQTITGSDPREAIFDTLCTDDSSEEAKTLTMDILTLAHTSTEAKGLSSESSASSDSPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGLHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSWSPGSDVTLLAEALVTVTNIEVINCSITEIETTTSSIPGASDTDLIPTEGVKASSTSDPPALPDSTEAKPHITEVTASAETLSTAGTTESAAPDATVGTPLPTNSATEREVTAPGATTLSGALVTVSRNPLEETSALSVETPSYVKVSGAAPVSIEAGSAVGKTTSFAGSSASSYSPSEAALKNFTPSETPTMDIATKGPFPTSRDPLPSVPPTTTNSSRGTNSTLAKITTSAKTTMKPPTATPTTARTRPTTDVSAGENGGFLLLRLSVASPEDLTDPRVAERLMQQLHRELHAHAPHFQVSLLRVRRG SEQ ID NO: 48 (MUC21) MKMQKGNVLLMFGLLLHLEAATNSNETSTSANTGSSVISSGASTATNSGSSVTSSGVSTATISGSSVTSNGVSIVTNSEFHTTSSGISTATNSEFSTASSGISIATNSESSTTSSGASTATNSESSTPSSGASTATNSDSSTTSSGASTATNSDSSTTSSEASTATNSESSTTSSGASTATNSESSTVSSRASTATNSESSTTSSGASTATNSESRTTSNGAGTATNSESSTTSSGASTATNSESSTPSSGAGTATNSESSTTSSGAGTATNSESSTVSSGISTVTNSESSTPSSGANTATNSESSTTSSGANTATNSDSSTTSSGASTATNSESSTTSSGASTATNSESSTTSSGASTATNSGSSTTSSGTSTATNSESSTVSSGASTATTSESSTTSSGASTATNSESSTVSSGASTATNSESSTTSSGANTATNSGSSVTSAGSGTAALTGMHTTSHSASTAVSEAKPGGSLVPWEIFLITLVSVVAAVGLFAGLFFCVRNSLSLRNTFNTAVYHPHGLNHGLGPGPGGNHGAPHRPRWSPNWFWRRPVSSIAMEMSGRNSGP SEQ ID NO: 49 (MUG HEG) MASPRASRWPPPLLLLLLPLLLLPPAAPGIRDPPPSPARRALSLAPLAGAGLELQLERRPEREPPPTPPRERRGPATPGPSYRAPEPGAATQRGPSGRAPRGGSAASESLHLPSSSSEFDERIAAFQTKSGTASEMGTERAMGLSEEWTVHSQEATTSAWSPSFLPALEMGELTTPSRKRNSSGPDLSWLHFYRTAASSPLLDLSSSSESTEKLNNSTGLQSSSVSQTKTMHVATVFTDGGPRTLRSLTVSLGPVSKTEGFPKDSRIATTSSSVLLSPSAVESRRNSRVTGNPGDEEFIEPSTENEFGLTSLRWQNDSPTFGEHQLASSSEVQNGSPMSQTETVSRSVAPMRGGEITAHWLLTNSTTSADVTGSSASYPEGVNASVLTQFSDSTVHSANAEDRTSGVPSLGTHTLATVTGNGERTLRSVTLTNTSMSTTSGEAGSPAAAMHQETEGASLHVNVTDDMGLVSRSLAASSALGVAGISYGQVRGTAIEQRTSSDHTDHTYLSSTFTKGERALLSITDNSSSSDIVESSTSYIKISNSSHSEYSSFFHAQTERSNISSYDGEYAQPSTESPVLHTSNLPSYTPTINMPNTSVVLDTDAEFVSDSSSSSSSSSSSSSSGPPLPLPSVSQSHHLFSSILPSTRASVHLLKSTSDASTPWSSSPSPLPVSLTTSTSAPLSVSQTTLPQSSSTPVLPRARETPVISFQTSTMTSFMTMLHSSQTADLKSQSTPHQEKVITESKSPSLVSLPTESTKAVTTNSPLPPSLTESSTEQTLPATSTNLAQMSPTFTTTILKTSQPLMTTPGTLSSTASLVTGPIAVQTTAGKQLSLTHPEILVPQISTEGGISTERNRVIVDATTGLIPLTSVPTSAKEMTTKLGVTAEYSPASRSLGTSPSPQTTVVSTAEDLAPKSATFAVQSSTQSPTTVSSSASVNSCAVNPCLHNGECVADNTSRGYHCRCPPSWQGDDCSVDVNECLSNPCPSTAMCNNTQGSFICKCPVGYQLEKGICNLVRTFVTEFKLKRTFLNTTVEKHSDLQEVENEITKTLNMCFSALPSYIRSTVHASRESNAVVISLQTTFSLASNVTLFDLADRMQKCVNSCKSSAEVCQLLGSQRRIFRAGSLCKRKSPECDKDTSICTDLDGVALCQCKSGYFQFNKMDHSCRACEDGYRLENETCMSCPFGLGGLNCGNPYQLITVVIAAAGGGLLLILGIALIVTCCRKNKNDISKLIFKSGDFQMSPYAEYPKNPRSQEWGREAIEMHENGSTKNLLQMTDVYYSPTSVRNPELERNGLYPAYTGLPGSRHSCIFPGQYNPSFISDESRRRDYF SEQ ID NO: 50 (MUC9) MWKLLLWVGL VLVLKHHDGA AHKLVCYFTN WAHSRPGPAS ILPHDLDPFL CTHLIFAFAS MNNNQIVAKD LQDEKILYPE FNKLKERNRE LKTLLSIGGW NFGTSRFTTM LSTFANREKF IASVISLLRT HDFDGLDLFF LYPGLRGSPM HDRWTFLFLI EELLFAFRKE ALLTMRPRLL LSAAVSGVPH IVQTSYDVRF LGRLLDFINV LSYDLHGSWE RFTGHNSPLF SLPEDPKSSA YAMNYWRKLG APSEKLIMGI PTYGRTFRLL KASKNGLQAR AIGPASPGKY TKQEGFLAYF EICSFVWGAK KHWIDYQYVP YANKGKEWVG YDNAISFSYK AWFIRREHFG GAMVWTLDMD DVRGTFCGTG PFPLVYVLND ILVRAEFSST SLPQFWLSSA VNSSSTDPER LAVTTAWTTD SKILPPGGEA GVTEIHGKCE NMTITPRGTT VTPTKETVSL GKHTVALGEK TEITGAMTMT SVGHQSMTPG EKALTPVGHQ SVTTGQKTLT SVGYQSVTPG EKTLTPVGHQ SVTPVSHQSV SPGGTTMTPV HFQTETLRQN TVAPRRKAVA REKVTVPSRN ISVTPEGQTM PLRGENLTSE VGTHPRMGNL GLQMEAENRM MLSSSPVIQL PEQTPLAFDN RFVPIYGNHS SVNSVTPQTS PLSLKKEIPE NSAVDEEA  SEQ ID NO: 51 (MUC18) MGLPRLVCAF LLAACCCCPR VAGVPGEAEQ PAPELVEVEV GSTALLKCGL SQSQGNLSHV DWFSVHKEKR TLIFRVRQGQ GQSEPGEYEQ RLSLQDRGAT LALTQVTPQD ERIFLCQGKR PRSQEYRIQL RVYKAPEEPN IQVNPLGIPV NSKEPEEVAT CVGRNGYPIP QVIWYKNGRP LKEEKNRVHI QSSQTVESSG LYTLQSILKA QLVKEDKDAQ FYCELNYRLP SGNHMKESRE VTVPVFYPTE KVWLEVEPVG MLKEGDRVEI RCLADGNPPP HFSISKQNPS TREAEEETTN DNGVLVLEPA RKEHSGRYEC QGLDLDTMIS LLSEPQELLV NYVSDVRVSP AAPERQEGSS LTLTCEAESS QDLEFQWLRE ETGQVLERGP VLQLHDLKRE AGGGYRCVAS VPSIPGLNRT QLVNVAIFGP PWMAFKERKV WVKENMVLNL SCEASGHPRP TISWNVNGTA SEQDQDPQRV LSTLNVLVTP ELLETGVECT ASNDLGKNTS ILFLELVNLT TLTPDSNTTT GLSTSTASPH TRANSTSTER KLPEPESRGV VIVAVIVCIL VLAVLGAVLY FLYKKGKLPC RRSGKQEITL PPSRKSELVV EVKSDKLPEE MGLLQGSSGD KRAPGDQGEK YIDLRH SEQ ID NO: 52 (p53 peptide)  QETFSDLWKLLPENN SEQ ID NO: 53 (p53 peptide)  DLWKLLPENNVLSPL SEQ ID NO: 54 (p53 peptide)  DDLMLSPDDIEQWFT SEQ ID NO: 55 (p53 peptide)  SPDDIEQWFTEDPGP SEQ ID NO: 56 (p53 peptide)  MPEAAPRVAPAPAAP SEQ ID NO: 57 (p53 peptide)  SVTCTYSPALNKMFC SEQ ID NO: 58 (p53 peptide)  YSPALNKMFCQLAKT SEQ ID NO: 59 (p53 peptide)  NKMFCQLAKTCPVQL SEQ ID NO: 60 (p53 peptide)  SQHMTEVVRRCPHHE SEQ ID NO: 61 (p53 peptide)  PQHLIRVEGNLRVEY SEQ ID NO: 62 (p53 peptide)  LRVEYLDDRNTFRHS SEQ ID NO: 63 (p53 peptide)  LDDRNTFRHSVVVPY SEQ ID NO: 64 (p53 peptide)  TFRHSVVVPYEPPEV SEQ ID NO: 65 (p53 peptide)  VVVPYEPPEVGSDCT SEQ ID NO: 66 (p53 peptide)  EPPEVGSDCTTIHYN SEQ ID NO: 67 (p53 peptide)  KKGEPHHELPPGSTK SEQ ID NO: 68 (p53 peptide)  HHELPPGSTKRALPN SEQ ID NO: 69 (p53 peptide)  KKLMFKTEGPDSD 

Example 2

Materials and Methods

Synthesis of O-glycopeptides: Chemoenzymatic Synthesis of LibrarySynthetic peptides: A MUC1 60-mer peptide (VTSAPDTRPAPGSTAPPAHG)_(n=3)representing three tandem repeats were kindly provided by CancerResearch UK. A 24-mer peptide derived from the C-terminal degeneratetandem repeats of MUC1 (AHGVTSAPDNRPALGSTAPPVHNV) was synthesized bySchafer-N. A MUC2 33-mer peptide (PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC)corresponding to 1.4 tandem repeat was synthesized as previouslydescribed (Sabbatini, Ragupathi et al. 2007). Eight chemicallysynthesized 21-mer MUC1 tandem repeat glycopeptides with single Tn or Tglycans were available. MUC4 peptides included: (PMTDTKTVTTPGSSFTA),(PGSSFTASGHSPSEIVPQD), (SEIVPQDAPTISAATTFAPA), (TTFAPAPTGNGHTTQAPTTA),(TTQAPTTALQAAPSSHD), (APSSHDATLGPSGGTSLSKT), (SLSKTGALTLANSVVSTP),(NSVVSTPGGPEGQWTSASAS), (TSASASTSPRTAAAMTHT), (AAAMTHTHQAESTEASGQT),(EASGQTQTSEPASSGSRTT), (PASSGSRTTSAGTATPSSS), (TATPSSSGASGTTPSGSEGI),(SGSEGISTSGETTRFSSN), (GETTRFSSNPSRDSHTT),(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP), (LPVTSLSSVSTGDTTPLPVTSPSSASTGH),(LPVTSPSSASTGHASPLLVTDASSASTGQ), (PLPVTSPSSASTGHASPLLVTDASSASTGQ),(STGDTLPLPVTDTSSV), (PVTYASSASTGDTTPLPVTDTSSVSTGHAT).

Peptides were synthesized at Peptides and Elephants, Gmbh, Germany orSchaefer-N, Copenhagen, Denmark.

Synthesis of recombinant gucin glycoprotein fragments in E. coli: N- orC-terminally 6×His and T7 tagged recombinant fragments of MUC2, MUC4,MUC5AC, MUC6, and MUC7 were produced in E. coli (See FIG. 1B). Genesequences were inserted into the bacterial expression vectors pET22(Novagen), pET28 (Novagen) or pET28 (minus), a modified pET 28 vectorwithout N-terminal tags. More than 10 bacterial strains were tested forexpression fidelity and efficacy of recombinant mucin protein fragments.Robustness of the system was tested using different expressionstrategies for mucin-like gene sequences. Expression yields varied from6 mg/l to 50 mg/l. Based on this, Rosetta2 (Novagen) was selected as theexpression host. Overnight cultures were diluted 1:100, and induced 4 hat 37° C. by the addition of IPTG to a final concentration of 0.1 mM.Cell lysates were nickel purified using NiNTA agarose (Qiagen) asdescribed by the manufacturer, and the eluted fractions were analyzed bySDS-PAGE on NuPAGE Bis-Tris 8-12% acryl amide gels (Novex) stained withCoomassie. Eluted fractions of recombinant mucin fragments were HPLCpurified before and after in vitro O-glycosylation (see below). Sampleswere diluted in 0.1% TFA (triflouroacetic acid), loaded onto a Zorbax300SB-C18 column mounted on an Agilent 1100 HPLC system, and eluted in a40-minute linear gradient from 0-90% acetonitrile. Eluted fractions werelyophilized and resuspended in water and mass confirmed by MALDI-TOFmass spectrometry. MALDI-TOF mass spectrometry was performed on aVoyager-DE™ PRO workstation (Applied Biosystems) using2,5-dihydroxybenzoic acid (Sigma) as matrix (Mirgorodskaya, Hassan etal. 1999).

Synthesis of O-glycopeptides: Peptides and recombinant fragments wereO-glycosylated in vitro using recombinant glycosyltransferases aspreviously described (Tarp, Sorensen et al. 2007) (Wandall, lrazoqui etal. 2007) Briefly, different polypeptide GalNAc-transferase isoformswere used to direct GalNAc O-glycan occupancies on peptides and theDrosophila Core-1 β3GalT, human Core-3 βGlcNAc-T and human ST6GalNAc-Iwere used to produce T, Core-3 and STn glycoforms. See FIG. 1A forstructures of glycopeptides. All glycopeptides were HPLC purified andcharacterized by MALDI-TOF.

Mucin O-glycopeptide array print and analysis: (Glyco)peptides andcontrol structures were printed on Schott Nexterion® Slide H or SchottNexterion® Slide H MPX 16 (Schott AG, Mainz, Germany). Quadruplicates ofall compounds were printed at 20, 5, and 1 μM in 150 mM sodium phosphatepH 8.5 with 0.005% CHAPS and printed on a BioRobotics MicroGrid IIspotter (Genomics Solution) with a 0.21 mm pitch using Stealth 3B MicroSpotting Pins (Telechem International Arraylt Division). After printing,slides were incubated for 1 h in a humidified hybridization chamber with75% relative humidity and stored until use at −20° C. Prior to use themicroarrays were blocked for 1 h with 25 mM ethanolamine in 100 mMsodium borate pH 8.5. Human sera serially diluted from 1:25-1:400 ormonoclonal antibodies (1 μg/ml or hybridoma supernatants) were incubatedin a closed container with gentle agitation for 1 h, washed three timesin PBS with 0.05% Tween-20 (PBS-T), followed by 1 h incubation withappropriate secondary antibodies. Human IgM and IgG antibodies weredetected with Cy3-conjugated goat anti-human IgG (Fc specific) and goatanti-human IgM (Sigma) diluted 1:5000 in PBS-T. For subclasscharacterization, biotinylated mouse anti-human IgM, IgG₁, IgG₂, IgG₃and IgG₄ (Sigma) were used and subsequently labelled withStreptavidin-DyLight547 (Pierce), both diluted 1:5,000 in PBS-T. Murinemonoclonal antibodies were detected with Cy3-conjugated goat anti-mouseIgM (μ chain specific) and goat anti-mouse IgG (H+L) (JacksonImmunoResearch Laboratories, Inc.) diluted 1:5000 in PBS-T. Afterincubation with secondary antibodies the slides were washed 3 times inPBS-T, and after the final wash, slides were rinsed shortly in H₂O,dried by centrifugation (200×g) and scanned in a ProScanArray HTMicroarray Scanner (PerkinElmer) followed by image analysis withProScanArray Express 4.0 software (PerkinElmer). Each spot were done in4 replicates and the mean value of relative fluorescence intensity (RFU)was used. A positive sample from the colorectal cancer group wasincluded on each experiment to confirm the reproducible of the assayallowing inter-experimental comparison. For comparison, slides werescanned with identical scanning parameters. Data were analyzed andplotted using Microsoft Excel or Graph Pad Prism software.

Monoclonal antibodies: Printing of the MUC1 glycopeptides was confirmedusing monoclonal antibodies against MUC1 (HMFG2)(5E10), T-MUC1 (1B9) and5E5 (Tn-MUC1). Additional mucin fragments were detected with anti-His(AD.1.10; Santa Cruz Biotechnology, USA; Sc-53073) and anti-T7antibodies (Novagen, USA; 69522) as well as carbohydrate-specificantibodies targeting Tn (1E3) and STn (3F1).

Human sera: Human colorectal cancer sera were obtained from Asterand,Inc., USA and from Hillerod Hospital. Sera from IBD patients werecollected from Herlev University Hospital, control sera were obtainedfrom healthy blood donors.

Quantification of serum MUC1: MUC1 capture ELISA was performed aspreviously described (Wandall 2009). Briefly, Immuno MaxiSorp F96 plates(Nunc) were coated with 1 μg/mL mAb HMFG2, blocked, and incubation withserially diluted sera. Amount of bound MUC1 was detected usingbiotinylated MUC1 specific antibody HMFG2.

Generation of monoclonal antibodies: Female Balb/c mice were immunizedwith 15Core-3-MUC1 60-mer glycopeptide, Tn-MUC4 20mer peptidePVTYASSASTGDTTPLPVTDTSSVSTGHAT and recombinant Tn-MUC4 all conjugated toKLH. Eye bleeds were collected 7 days after the third immunisation andsera tested by ELISA, with the Core-3/Tn-MUC2 glycopeptide serving asnegative control or by immunocytochemistry. Three days after the fourthimmunisation, spleen cells from one mouse were fused with NS1 myelomacells (Kohler and Milstein 1975). Hybridomas specific to the antigens ofinterest were cloned by limiting dilution at least three times.

Immunocytochemistry and immunohistochemistry: 6-10 μm tissue sectionswere fixed for 5 min in ice-cold acetone or 4% paraformaldehyde. Fixedcells were incubated 2 hours at room temperature or overnight at 4° C.with undiluted mAb supernatants, followed by incubation for 45 min atroom temperature with fluorescein isothiocyanate-conjugated rabbitanti-mouse immunoglobulins in 1:100 dilution with 0.1% BSA (Dako).Slides were mounted in glycerol-containing p-phenylenediamine andexamined in a Zeiss fluorescence microscope.

Cellular immune response: Peripheral blood mononuclear cells (PBMCs)were isolated from heparin anti-coagulated blood of patients with latestage (III-IV) colo-rectal cancer using Lymphoprep (Nycomed Pharma AS,Oslo, Norway). Cells were then cultured in X-VIVO 15 medium (Invitrogen)together with 2% heat inactivated human AB serum (Valley Biomedical,Winchester, USA) in round bottom microculture plates (Nunc, Roskilde,Denmark). Cells were cultured as 2*10⁵ cells/well and stimulated withpeptide pools with 10 peptides in each pool (all from Schafer-N,Copenhagen, Denmark). The concentration of each peptide was 10 mg/ml andthe peptide pools were 15-mer peptides with 10 amino acid overlapping,which allowed to test for reactivity against peptides representing thewhole MUC1 peptide backbone. 50 IU/ml IL-2 (Proleukin, Chiron, TheNetherlands) was added at day 1 and cells were harvested at day 9 andrestimulated with the same peptide pools or without peptide as anegative control in an IFN-g ELISPOT assay (Svane, Pedersen et al. 2004)in order to identify MUC-1 specific CD4+ or CD8+ T cells.

Results

Assembly of the Mucin O-Glycopeptide Array

The employed strategy was based on chemo-enzymatic synthesis of largeglycopeptides, either based on synthetic peptides or large mucinfragments produced in e-coli, and use micro-array technology to identifythe most promising targets, followed by their deconvolution byoverlapping glycopeptides.

We first generated a comprehensive glycopeptide mucin array coveringdomains from MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7 carrying cancerassociated glycans Tn, Sialyl-Tn and truncated Core3. Next targetsselective identified The MUC1 tandem repeat was represented by 60mersynthetic peptides as well as recombinant protein. In order to covermultiple potential epitopes in the large mucin proteins we producedrecombinant fragments of MUC2, MUC4, MUC5AC, MUC6, and MUC7 by a simpleand robust E. coli expression system. The mucin fragments wereglycosylated with a combination of recombinant GalNAc-transferases(GalNAc-T1-4) yielding glycosylation products with high density ofGalNAc addition comparable to what is expected in cancer cells. Furtherelongation of the Tn-glycoforms with sialyltransferase ST6GalNAc-T1 (tosynthesize STn) and β3GlcNAc-T6 (to synthesize Core-3) yielded nearcomplete glycosylation of all fragments as verified by SDS-PAGE andMALDI-TOF spectrometry (FIG. 7). The integrity of the printed mucinstructures was verified by consistent labelling with mAbs to MUC1(HMFG2), T7-tag, Tn (GalNAc-α-S/T)(1E3/5F4), and STn (NeuAcα2,6GalNAc)(FIG. 1D. Some residual Tn reactivity was detected in sialylated andCore-3 elongated glycosylation products, which was expected due to thevery high density of Tn structures on the recombinant proteins.Purification of each glycosylation product followed by re-glycosylationwith ST6GalNAc-1 or Core-3 synthase did not prevent such Tn exposure,excluding that the incomplete Tn-elongation was due to impurities andaccumulation of by-products during in vitro glycosylation. Qualitycontrol of the microarrays was ensured by the inclusion a positivecancer sample as standard in each array analysis. The CV (coefficient ofthe variation) value differed between target compounds. For the targetsof interest, however, it was less than 10%.

IgG Auto-antibodies Recognizing Cancer Associated Glycans in Combinationwith MUC1 Peptide Backbone as Biomarkers in Serum

The development of the glycopeptide mucin array allowed us to test forthe presence of auto-antibodies in newly diagnosed patients withcolorectal cancer (n=58) and healthy controls n=50 (FIG. 2). To oursurprise we did not detect any significant IgG signature unambiguouslyidentifying colorectal cancer patients from healthy individuals with theeither non-glycosylated or glycosylated (Tn-, STn-, and truncatedCore-3) recombinant mucin fragments (MUC2, MUC4, MUC5AC, MUC6, and MUC7)(FIG. 8). In contrast specific IgG auto-antibodies against Tn-MUC1,STn-MUC1 or truncated Core-3-MUC1 glycopeptide epitopes were identifiedin colorectal cancer patients (FIGS. 2 and 3A), with none or very lowlevel of reactivity in healthy individuals. The reactivity with Tn-MUC1,STn-MUC1 or truncated Core-3-MUC1 glycopeptide epitopes were veryhomogeneous and the mean IgG reactivity expressed as fluorescenceintensity was used to define the cut-off level for each glyco-peptidetarget. A serum sample was determined as positive if the fluorescentreactivity with a given glyco-peptide target was higher than three timesthe SD above the mean of the values obtained with sera from the healthygroup. By this definition 74.1% of colorectal cancer patients hadcirculating antibodies towards either non-glycosylated, Tn, STn-, orCore3-glycosylated MUC1 (FIGS. 2 and 3, Table I). The most selective ofthe three antibody targets was STn-MUC1, which detected 56.7% (33/58)cancer patients, while Tn-MUC1 detected 39.7 (23/58) and Core3-MUC1antibodies 44.8% (26/58). For comparison 0-2% healthy individuals testedpositive on the different glycoforms of MUC1. An extra set of controls(n=50) were analyzed using these parameters. 2% (1/50) of these controlstested positive for STn-MUC1 antibodies, (1/50) for Tn-MUC1, and (1/50)for Core-3-MUC1 antibodies (FIG. 2,3A, Table I). Importantly theseresults were confirmed with both synthetic peptides covering the MUC1repeat as well as recombinant MUC1. The induced antibodies did notcross-react with Tn or STn haptens as evidenced by lack of reactivitywith other Tn and STn-glycopeptides. In sera from a few cancer patients(4/58; 6,9%) minimal reactivity with non-glycosylated MUC1 was detected,similar to the levels previously reported in breast cancer (vonMensdorff-Pouilly, Petrakou et al. 2000). Tn- and STn-MUC1 glycopeptideswith at least two O-glycans in the immunodominant —GSTAP-epitope, i.e. 9and 15 Tn-MUC1 (with two O-glycans in the —GSTAP-epitope) detectedsubstantially more cancer patients than 6Tn-MUC1 (with one O-glycan inthe —GSTAP-epitope). In particular glycopeptides with five glycans perrepeat (VTSAPDTRPAPGSTAPPAHG) detected a higher number of colorectalcancer patients than glycoforms with three glycans per repeat(VTSAPDTRPAPGSTAPPAHG) (FIG. 2, Table I). This indicates that aconsiderable number of patients had auto-antibodies directed against theglycosylated PDTR epitope and/or the additional epitope formed by twoGalNAc in the —VTS-region of the MUC1 repeat. To define the specificityof the cancer induced auto-antibodies we analyzed serum reactivity withMUC1 peptides carrying GalNAc, STn, and Core-3 at specific glycosylationsites (Table I). The majority of the cancer patients demonstrated theirmain reactivity with —GST-epitope (82% n/n), which corresponds with thefinding that 15 STn/Tn/Core-3-MUC1 60mer peptide was the best targetantigen (Table I). Some patients demonstrated polyclonal responses withadditional reactivity against the glycosylated -PDTR-epitope (14%) and—VST-epitope (4%).

Glycoform Specificity of MUC1 Auto-antibodies Discriminates BetweenColorectal Cancer and Inflammatory Bowel Disease

An inherent problem with many cancer markers identified to date is theirlack of discrimination between cancer disease and inflammatory lesionscausing benign diseases to be identified as cancer. Based on variationsin glycan expression by cancer and inflammatory lesions we hypothesizedthat glycoform specificities of MUC1 auto-antibodies could distinguishbetween patients with colorectal cancer and chronic inflammatory boweldisease (IBD). Sera from patients diagnosed with Ulcerative Colitis orCrohn's disease in either active or remission state were thereforeincluded in the study for comparison. In accordance with our hypothesisthe cancer specific STn-MUC1 antibody response detected in 56,9% (33/58)of the colorectal cancer patients was only detected in 5.9% of IBDpatients (FIG. 2,3 and Table I). This is in agreement with previousfindings that tissue expression of the STn epitope is solely associatedwith dysplasia and colorectal cancer in IBD patients (Itzkowitz, Bloomet al. 1990). In contrast we found significant signals forauto-antibodies to Core-3-MUC1 28.2%) (FIG. 2A and Table I). Whensearching for the existence of auto-antibodies for the remaining mucinfragments we observed a higher reactivity to the MUC4, MUC5AC and MUC6in the IBD population compared with healthy and cancer patients,although this was not-significant.

Characterization of MUC1 Glyco-peptide Auto-antibodies

In order to verify the selective nature of the identifiedauto-antibodies and eliminate cross-reactivity of the glyco-peptideantibodies inhibition studies were performed. The cancer specific serumreactivity to STn-MUC1 and Core-3-MUC1 was selectively inhibited by 40μg/mL of the respective MUC1 glycopeptides STn-MUC1 and Core3-MUC1 (FIG.3B). Importantly, the reactivity of other mucins and glycoforms was leftuninhibited. Only slight inhibition of the Core3-MUC1 reactivity wasseen with free carbohydrate GlcNAc confirming that the peptide contextof the carbohydrate structure plays an essential role. The selectivenature of the cancer generated glycopeptides-MUC1 antibodies wasadditionally verified by pull-down assays with recombinant Tn-, STn,Core-3-MUC1 coupled to Dynabeads® (FIG. 9). The affinity purified serumIgG antibodies selectively reacted with the respective glycoform ofMUC1, while the reactivity was diminished in the depleted serum. Inaccordance with previous findings, IgM antibodies purified onCore-3-MUC1-beads revealed hapten specificity reacting with all mucinscarrying Core3. We finally determined the subclass of a subset of thecirculating auto-antibodies in cancer patients reactive with STn- andCore3-MUC1. Interestingly these were mainly (68%) of the IgG₂ subclassalong with 23% of the patients having IgG₃ and 9% having IgG₁/_(3/4).

To explain the large interpersonal variance in auto-antibody levels wenext examined the presence of measurable cellular response against MUC1in a separate cohort of patients. For this purpose we used peptide poolsthat represented T cell epitopes from the whole peptide backbone ofMUC1. However, no MUC1 specific CD4+ or CD8+ T cells could beidentified. Another possibility for interpersonal variations could bevariable presence of circulating antigen. To correlate the presence ofcirculating MUC1 in cancer patients with the presence of auto-antibodieswe next employed a capture ELISA strategy using the mAb HMFG2recognizing all-glycoforms of MUC1. Although, various MUC1 glycoformssecreted from cancer cell lines (T47D) were readily detected by thismethod (Wandall 2009), the inventors could not detect any circulatingMUC1 in colorectal cancer patients. This is in accordance with the lackof MUC1 detection using the commercial available CA 15-3 assay (Gebauer,Jager et al. 1998), and suggests that most secreted MUC1 is selectivelycleared from the circulation.

IgA Autoantibody Signatures Against Tn-MUC4 Aid Selective Detection ofColorectal Cancer

Because of the large quantities of IgA produced in the colon we nextextended our analysis to test for the presence of auto-antibodies of theIgA subclass. The rationale for this approach was the knowndown-regulation of epithelial transcytosis of polymeric

IgA in colon carcinomas mediated by the polymeric immunoglobulinreceptor (plgR) and loss of cellular polarity in the carcinoma cells.The combined effect of these events would be expected to increasecirculating IgA specific for relevant cancer targets (Kaetzel 2005)(Baseler, Maxim et al. 1987). In accordance with the hypothesis, wedetected increased levels of IgA auto-antibodies targeting the GalNAcglycosylated recombinant MUC4 fragment (Tn-MUC4) or non-glycosylatedMUC4 in 75% of colorectal cancer. This finding was MUC4 specific. Incontrast IgA auto-antibody reactivity to membrane mucin MUC1 andsecreted mucins MUC2, MUC5AC, MUC6, and MUC7 could not be used todiscriminate between colon cancer patients and healthy individuals. Toevaluate if the IgA auto-antibody responses to MUC4 were due to a singleimmunodominant epitope or the collective reactivity of a polyclonalimmune-responses, we analyzed overlapping glycopeptides covering therecombinant fragment of MUC4 with the inclusion of an additional set of20mer MUC4 peptides covering the MUC4 tandem repeat area. The immuneresponse against MUC4 had a polyclonal nature. However, up to 30% of thecolorectal cancer patients had IgA autoantibodies to a single MUC4tandem repeat peptide (TRM4-5; PVTYASSASTGDTTPLPVTDTSSVSTGHAT), with themajority of these patients having glycopeptide specific responses toGalNAc-glycosylated MUC4 tandem repeat peptide (FIG. 4, C). Apart fromthe immunodominant Tn-TRM4-5 glycopeptide, each cancer patientregognised different peptide and glyco-peptide epitopes among theremaining MUC4 targets. In order to compile results obtained with allMUC4 peptides, the values obtained for each target were expressed as thenumber of SD above the mean values from healthy patients. Thereby, amultiplex result was constructed, which demonstrated that 79.3% ofcancer patients had detectable IgA MUC4 antibodies with cut-off valueselected to be five times the SD of the mean of values from healthypatients to ensure high specificity. Importantly, the level of IgAreactivity was much lower in patients with inflammatory bowel disease(FIG. 4). We next tested IgA levels against MUC4 in serum from patientswith other cancer than colorectal (prostate, ovarian, breast). Asignificantly portion of the patients had IgA auto-antibodies againstepitopes outside the tandem repeat, but only little reactivity to thetandem-repeat region (7.1%) compared with the colorectal cancer group(55.1%).

Generation of Glycopeptide Specific Antibodies to MUC1 and MUC4Glycopeptides

Two of the identified immodominant MUC1 glycopeptides (Tn-MUCI andSTn-MUC1) has previously been shown to override tolerance in humans andhumanized mice with the generation of potent monoclonal antibodies (5E5and 2D9) specific for combined glycopeptide epitopes (Sorensen, Reis etal. 2006) (Tarp, Sorensen et al. 2007). In the present study we testedif the novel identified immunodominant glycopeptides Core3-MUC1 and thetwo different Tn-MUC4 targets (Tn-recMUC4 and Tn-MUC4TR5) inducedimmune-responses in wild type mice enabling generation of glycopeptidesspecific monoclonal antibodies. 60mer MUC1-Core3 glycopeptide withcomplete 0-glycan occupancy and conjugated to KLH elicited strongpolyclonal antibody response in Balb/c mice reacting with fullyglycosylated Core3-MUC1 as demonstrated by ELISA (FIG. 5). A monoclonalantibody was produced with specificity for MUC1 carrying the Core-3structure in T in -PDTR- in the MUC1 tandem repeat as analyzed by ELISAand array presenting mucins with cancer associated glycans. Reactivitywas detected with Sialylated-Core3 as demonstrated by array analysis(FIG. 5C-E). No cross reactivity was detected with non-Core3-basedstructures or with other mucins carrying Core-3. In the same way, wetested the immunogenicity of Tn-MUC4rec and Tn-MUC4TR5-KLH. In bothcases a prominent immuneresponse specific for Tn-MUC4 was generated inBalb/c mice (FIG. 5, Panel I). A monoclonal antibody 6E3 was generatedusing Tn-MUC4rec as immunogen. Elisa and array analysis demonstratedstrong reactivity with Tn-MUC4 (FIG. 51-L). No reactivity was seen withnon-glycosylated peptide, while some cross-reactivity was noted withTn-MUC1. Both the monoclonal antibody 6E3 and the polyclonal sera frommice immunized with Tn-MUC4TR stained HT29 and LSC cells known toexpress Tn-MUC4 due to disrupted cosmc and hence T-synthase function. Noreaction was seen with cells not expressing MUC4. These findings confirmthe immunogenic nature of the glycopeptides identified as targets in thescreen for autologous antibodies and provide valuable tools for studyingthe expression of such glycopeptide epitopes in human cancer andinflammatory lesions.

Tissue Expression of Immunodominant MUC1 and MUC4 Glycopeptide Epitopesin Colorectal Cancer and Inflammatory Bowel Disease

Using the developed glycopeptide specific antibodies we next tested theexpression of Tn/STn-MUC1, Core3-MUC1, Tn-MUC4 in healthy tissue, tissuefrom inflammatory bowel patients and cancer patients. Examination of 25cases of colorectal adenocarcinomas for the expression of Tn-, STn, andCore-3 MUC1 was performed with the well-characterized mAbs HMFG2, 5E5and 2D9 with specificity for non-glycosylated MUC1, Tn- and STn-MUC1respectively (Sorensen, Reis et al. 2006; Tarp, Sorensen et al. 2007).Core-3-MUC1 and Tn-MUC4 expression was analyzed with the novelCore3-MUC1, Tn-MUC4 mAbs (5C10, 6E3) respectively. 92% (23/25) of thecancer cases were positive, with between 20-90% of the cancer cellsstaining bright positive. Intensive labelling of intracellularstructures as well as the luminal surface of cancer cells were seen withmAbs HMFG2, 5E5, 5C10, and 2D9 verifying the presentation of largeamounts of Tn-, STn, and Core3-MUC1 on cancer cells. Importantly,apparent healthy neighbouring tissue had substantial lower expressionlevels, although a supranuclear staining pattern were seen in mostcells.

ADCC 6E3

Peripheral blood mononuclear cells (PBMC) were isolated from the bloodof healthy donors by density centrifugation with Lymphoprep (NycomedPharma Diagnostics, Oslo, Norway). The blood was diluted 1:2 in PBS with0.5% FBS and was gently poured down the side of 50 ml tubes containing15 ml Lymphoprep. After centrifugation at 2000 rpm in 20 min, the layercontaining PBMCs was transferred to a new tube, washed, and resuspendedin RPMI 1640 before eosin staining for evaluation of viability.

ADCC were tested using a standard 51Cr release assay. Target cells wereloaded with 100 μCi 51Cr (PerkinElmer) for 1 h at 37° C., and washed inculture media×5, and plated at 1×104 per well in a 96-well flat-bottomplate before incubation for 1 hour with antibody before addition ofeffector cells. After 4 hours of incubation at 37° C., 30 μl ofsupernatant was removed from each well and plated on a LumaPlate-96,dried down, and counted on a Packard's TopCount®.

The results demonstrated in FIG. 13 are expressed as the percentage ofspecific release calculated by following equation: (experimentalrelease−spontaneous release×100)/(maximal release−spontaneous release),where the spontaneous release represents the mean cpm for target cellsincubated without antibody and immuneeffectors, and the maximal releaserepresents the mean cpm for target cells incubated with 30% ethanol.

P53 Array Print and Analysis.

15 mer peptides with 10 amino acid overlap representing the whole p53protein backbone and control structures were printed on SchottNexterion® Slide H or Schott Nexterion® Slide H MPX 16 (Schott AG,Mainz, Germany). Quadruplicates of all compounds were printed at 200 and50 μM in 150 mM sodium phosphate pH 8.5 with 0.005% CHAPS and printed ona BioRobotics MicroGrid II spotter (Genomics Solution) with a 0.21 mmpitch using Stealth 3B Micro Spotting Pins (Telechem InternationalArraylt Division). After printing, slides were incubated for 1 h in ahumidified hybridization chamber with 75% relative humidity and storeduntil use at −20° C. Prior to use unspotted slide areas were blocked for1 h with 25 mM ethanolamine in 100 mM sodium borate pH 8.5. Human seraserially diluted 1:25 and were incubated in a closed container withgentle agitation for 1 h, washed three times in PBS with 0.05% Tween-20(PBS-T), followed by 1 h incubation with appropriate secondaryantibodies. Human IgG antibodies were detected with Cy3-conjugated goatanti-human IgG (Fc specific) diluted 1:5000 in PBS-T. After incubationwith secondary antibodies the slides were washed 3 times in PBS-T, andafter the final wash, slides were rinsed shortly in H₂O, dried bycentrifugation (200×g) and scanned in a ProScanArray HT MicroarrayScanner (PerkinElmer) followed by image analysis with ProScanArrayExpress 4.0 software (PerkinElmer). Each spot were done in 4 replicatesand the mean value of relative fluorescence intensity (RFU) was used.For comparison, slides were scanned with identical scanning parameters.Data were analyzed and plotted using Microsoft Excel or GraphPad Prismsoftware. A total of 78 15mer peptides were printed. 18 peptides p53peptides (number: 4, 5, 9, 10, 14, 25, 26, 27, 34, 39, 41, 42, 43, 44,45, 58, 59, and 78) had had sensitivity over 10% with specificity 95%.19 out of 58 colorectal cancer patients had autoantibodies to peptide34. Combining the p53-34 with MUC1STn increased the sensitivity from 57%to 72%; a combination of p53-34, p53-44, and MUC1 STn increased thesensitivity to 79% with a specificity of 94%. The results aredemonstrated in FIG. 15.

TABLE I IgG Auto-antibodies to selected MUC1 glycopeptides Percentage ofhealthy controls, IBD patients and colorectal cancer patients withauto-antibodies to MUC1 glycopeptides. A positive test is defined as 3standard deviation over the mean of the healthy controls. Combinedresults with two or three glycopeptides are also shown. GlycopeptideControls IBD CRC MUC1 4% (2/50) 2.6% (1/39) 6.9% (4/58) MUC1 6Tn 2%(1/50) 5.1% (2/39) 20.7% (12/58) MUC1 9Tn 2% (1/50) 5.1% (2/39) 20.7%(12/58) MUC1 15Tn 2% (1/50) 15.4% (6/39)  39.7% (23/58) MUC1 9Core3 2%(1/50) 17.9% (7/39)  27.6% (16/58) MUC1 15Core3 0% (1/50) 20.5% (8/39) 44.8% (26/58) MUC1 9STn 2% (1/50) 7.7% (3/39) 41.4% (23/58) MUC1 15STn2% (1/50) 10.3% (4/39)  56.9% (33/58) MUC1 9C3 or 15C3 2% (1/50) 28.2%(11/39) 44.8% (26/58) MUC1 STn or C3 4% (2/50)   33% (13/39) 63.8%(37/58) MUC1 Tn or STn or 8% (4/50) 38.5% (15/39) 74.1% (43/58) C3

TABLE II IgA Auto-antibodies to selected MUC4 glycopeptides Percentageof the healthy controls, IBD patients, colorectal cancer patients, and acombined group of prostate, ovarian and breast cancer patients with IgAauto- antibodies to different MUC4 glycopeptides. A positive test isdefined as 5 standard deviation over the mean of the healthy controls.Combined results with two or more glycopeptides are also shown.Glycopeptide Prostate, Ovarian and Controls IBD CRC Breast cancer MUC40% 2.6% (1/39) 29.3% (17/58) 3.6% (1/28) MUC4 Tn 0% 7.7% (3/39) 37.9%(22/58) 7.1% (2/28) MUC4 non 0% 7.7% (3/39) 55.1% (32/58) 7.1% (2/28)glycosylated or Tn MUC4s fragments 0% 17.9% (7/39)  41.4% (24/58) 35.7%(10/28) MUC4s fragments Tn 0% 7.7% (3/39) 29.3% (17/58) 57.1% (16/28)All Tn-MUC4 0% 7.7% (3/39) 53.4% (31/58) 57.1% (16/28) recMUC4s 0% 2.6%(3/39) 3.4% (2/58) 7.1% (2/28) recMUC4s Tn 0% 2.6% (3/39) 13.8% (8/58) 32.1% (9/28)  MUC1Tn, STn, C3 8% 43.6% (17/39) 87.9% (51/58) MUC4 Tn, TRMUC1STn + MUC4 2% 15.4% (6/39)  82.8% (48/58)

TABLE III Legend to glycopeptide sequences of Table II MUC4(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP) (LPVTSLSSVSTGDTTPLPVTSPSSASTGH)(LPVTSPSSASTGHASPLLVTDASSASTGQ) (STGDTLPLPVTDTSSV)(PVTYASSASTGDTTPLPVTDTSSVSTGHAT) MUC4 Tn(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ) (S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*) MUC4 non(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP) glycosylated or(LPVTSLSSVSTGDTTPLPVTSPSSASTGH) Tn (LPVTSPSSASTGHASPLLVTDASSASTGQ)(STGDTLPLPVTDTSSV) (PVTYASSASTGDTTPLPVTDTSSVSTGHAT)(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ) (S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*) MUC4sPMTDTKTVTTPGSSFTA (SEQ ID NO: 3), fragmentsPGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8),SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10),TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12),EASGQTQTSEPASSGSRTT (SEQ ID NO:13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14),TATPSSSGASGTTPSGSEGI (SEQ ID15 NO: 15),GSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17)MUC4s PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO: 3), fragments TnPGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4),S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD (SEQ ID NO: 7),APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO: 8)S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10),T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT* (SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 No: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16),GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17) All Tn-MUC4(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ) (S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*)(PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT2FAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*)PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO: 3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4),S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD (SEQ ID NO: 7),APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO: 8)S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10),T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT* (SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16),GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17) Recombinant(PMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGN MUC4sGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFSSNPSRDS HTT) Recombinant(PMT*DT*KT*VT*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*Z MUC4s TnFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*) MUC1Tn,STn,C3(MUC1STn) MUC4 Tn VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHGVT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHGVT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG MUC4(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP) (LPVTSLSSVSTGDTTPLPVTSPSSASTGH)(LPVTSPSSASTGHASPLLVTDASSASTGQ) (STGDTLPLPVTDTSSV)(PVTYASSASTGDTTPLPVTDTSSVSTGHAT)(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ) (S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*)(PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*)PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO: 3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4),S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ ID NO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD (SEQ ID NO: 7),APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO 8)S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ ID NO: 10),T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT* (SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16),GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17) MUC1STn+ MUC1STn MUC4VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG MUC4(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP) (LPVTSLSSVSTGDTTPLPVTSPSSASTGH)(LPVTSPSSASTGHASPLLVTDASSASTGO) (STGDTLPLPVTDTSSV)(PVTYASSASTGDTTPLPVTDTSSVSTGHAT)(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ) (S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*)

The invention claimed is:
 1. A Tn-MUC4 binding polypeptide comprisingthe six complementarity determining regions (CDRs) of the monoclonalantibody produced by the cell line 6E3 deposited with the EuropeanCollection of Authenticated Cell Cultures (ECACC) under accession number09120103.
 2. The Tn-MUC4 binding polypeptide of claim 1, which ismonospecific.
 3. The Tn-MUC4 binding polypeptide of claim 2, which ismonovalent.
 4. The Tn-MUC4 binding polypeptide of claim 1, which ismultispecific.
 5. The Tn-MUC4 binding polypeptide of claim 4, which isbispecific.
 6. The Tn-MUC4 binding polypeptide of claim 5, which iscapable of specifically binding a cluster of differentiation (CD)protein in addition to Tn-MUC4.
 7. The Tn-MUC4 binding polypeptide ofclaim 6, wherein the CD protein is CD64 or CD89.
 8. The Tn-MUC4 bindingpolypeptide of claim 1, which is an IgA, IgG, IgD, IgE or IgM antibody.9. The Tn-MUC4 binding polypeptide of claim 8, which is an IgG antibody.10. The Tn-MUC4 binding polypeptide of claim 1, which is the monoclonalantibody produced by the cell line 6E3deposited with the ECACC underaccession number
 09120103. 11. The Tn-MUC4 binding polypeptide of claim1, which comprises an antibody fragment.
 12. The Tn-MUC4 bindingpolypeptide of claim 11, wherein the antibody fragment is a Fab, Fab′,F(ab′)₂, Fv, or scFv.
 13. The Tn-MUC4 binding polypeptide of claim 11,wherein the antibody fragment is an antigen binding fragment of themonoclonal antibody produced by the cell line 6E3deposited with theECACC under accession number
 09120103. 14. The Tn-MUC4 bindingpolypeptide of claim 13, wherein the antibody fragment is a Fab, Fab′,F(ab′)₂, or Fv fragment of the monoclonal antibody produced by the cellline 6E3 deposited with the ECACC under accession number
 09120103. 15. Aconjugate comprising the Tn-MUC4 binding polypeptide of claim 1conjugated to an imaging agent.
 16. The conjugate of claim 15, whereinthe imaging agent is a fluorescent label, an antibody, a small molecule,a peptide, a transition metal ion, a lanthanide ion, or an actinide ion.17. The conjugate of claim 16, wherein the imaging agent is an ion ofHf, Ho, or Gd.
 18. A conjugate comprising the Tn-MUC4 bindingpolypeptide of claim 1 conjugated to a toxin.
 19. The conjugate of claim18, wherein the toxin is a small molecule, a metal, a metal ion, a smallinorganic molecule, a protein, a peptide, a glycopeptide, RNA, DNA, orsiRNA.
 20. A conjugate comprising the Tn-MUC4 binding polypeptide ofclaim 1 conjugated to a chemotherapeutic agent.
 21. The conjugate ofclaim 20, wherein the chemotherapeutic agent comprisesaminoglutethimide, aminopterin, azathioprine, bleomycin sulfate,bulsulfan, carboplatin, carminomycin, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclosporine, cytarabidine, cytosinearabinoside, cytoxin dacarbazine, dactinomycin, daunomycin,daunorubicin, doxorubicin, an esperamicin, etoposide, fluorouracil,ifosfamide, interferon-α, lomustine, melphalan, mercaptopurine,methotrexate, mitomycin C, mitotane, mitoxantrone, procarbazine HCI,taxol, docetaxel, teniposide, thioguanine, thiotepa, vinblastinesulfate, vincristine sulfate or vinorelbine.
 22. A method for detectinga Tn-MUC4-expressing cancer in a subject comprising: (a) contacting asample from the subject with the Tn-MUC4 binding polypeptide of claim 1;(b) removing unbound sample from the Tn-MUC4 binding polypeptide orunbound Tn-MUC4 binding polypeptide from the sample; and (c)characterizing the binding of the Tn-MUC4 binding polypeptide to thesample, wherein binding of the Tn-MUC4 binding polypeptide to the sampleis indicative of cancer in the subject.
 23. The method of claim 22,wherein the cancer is colorectal cancer.
 24. A method of treating cancerin an individual in need thereof, comprising administering to theindividual the Tn-MUC4 binding polypeptide of claim
 1. 25. The method ofclaim 24, wherein the cancer is colorectal cancer.
 26. A method oftreating cancer in an individual in need thereof, comprisingadministering to the individual the conjugate of claim
 20. 27. Themethod of claim 26, wherein the cancer is colorectal cancer.
 28. A cellof the hybridoma cell line 6E3, which is deposited with the ECACC underaccession number 09120103.